Expression of nuclear receptor co­activator 7 protein is associated with poor prognosis of breast cancer.

Nuclear receptor coactivator 7 (NCOA7) is an estrogen receptor binding protein. Its role in breast cancer progression has so far remained elusive. The present study aimed to determine the expression levels of NCOA7 in breast tumor samples and confirmed its potential utility as a breast cancer prognostic biomarker. The expression of NCOA7 was detected by immunohistochemical staining in 241 breast cancer tumor samples and 163 adjacent normal tissue samples. The association of NCOA7 expression with the clinicopathological characteristics and overall survival were statistically analyzed. Cell proliferation was determined by Cell Counting Kit-8 and colony-formation assays. Cell migration was detected using wound-healing and Transwell assays. NCOA7 was positively expressed in 44% of breast tumor tissues. The expression of NCOA7 was positively associated with tumor size (T-stage; P=0.005) and lymph node metastasis (N-stage; P=0.008). Additional statistical analysis indicated that the expression of NCOA7 was associated with patient age, tumor size and lymph node metastasis in patients with triple-negative breast cancer (TNBC) compared with that in patients with non-TNBC. The overall survival of patients with NCOA7-positive breast cancer was significantly lower than that of patients with NCOA7-negative breast cancer (P=0.006). Among the patients with lymph node metastasis, the overall survival was reversely associated with the expression of NCOA7 (P=0.042). Furthermore, knockdown of NCOA7 expression in breast cancer T47D and MCF7 cells significantly inhibited both cell proliferation and migration, suggesting that this protein may exert a role in driving breast cancer progression. Taken together, these results indicate that the expression of NCOA7 is associated with poor prognosis of breast cancer and suggest that this protein may be a driver for metastasis and a potential therapeutic target for advanced breast cancer.

Activation of E3 ubiquitin ligase WWP2 by non-receptor tyrosine kinase ACK1.

WW domain containing E3 ubiquitin protein ligase 2 (WWP2) is a member of the NEDD4 E3 ubiquitin ligase family. WWP2 ligase activity is regulated by the 2, 3-linker auto-inhibition. Tyrosine phosphorylation of the 2, 3-linker was identified as an activating means for releasing the auto-inhibition of WWP2. However, the tyrosine kinase (TK) for the phosphorylation and activation remains unknown. In this report, we have found that non-receptor TK ACK1 binds to the WW3 domain of WWP2 and phosphorylates WWP2. ACK1 phosphorylates WWP2 at the 2, 3-linker and partially activates the ubiquitination ligase activity. Unexpectedly, tyrosine phosphorylation of the 2, 3-linker seems not a major mode for activation of WWP2, as ACK1 causes much higher activation of the 2, 3-linker tyrosine phosphorylation defective mutants of WWP2 than that of wild-type WWP2. Furthermore, epidermal growth factor (EGF) stimulates tyrosine phosphorylation of WWP2 and this EGF-stimulated phosphorylation of WWP2 is mediated by ACK1. Finally, knockdown of WWP2 by shWWP2 inhibits the EGF-dependent cell proliferation of lung cancer A549 cells, suggesting that WWP2 may function in the EGFR signaling in lung cancer progression. Taken together, our findings have revealed a novel mechanism underlying activation of WWP2.

Emerging roles of the HECT E3 ubiquitin ligases in gastric cancer.

Gastric cancer (GC) is one of the most pernicious gastrointestinal tumors with extraordinarily high incidence and mortality. Ubiquitination modification of cellular signaling proteins has been shown to play important roles in GC tumorigenesis, progression, and prognosis. The E3 ubiquitin ligase is the crucial enzyme in the ubiquitination reaction and determines the specificity of ubiquitination substrates, and thus, the cellular effects. The HECT E3 ligases are the second largest E3 ubiquitin ligase family characterized by containing a HECT domain that has E3 ubiquitin ligase activity. The HECT E3 ubiquitin ligases have been found to engage in GC progression. However, whether HECT E3 ligases function as tumor promoters or tumor suppressors in GC remains controversial. In this review, we will focus on recent discoveries about the role of the HECT E3 ubiquitin ligases, especially members of the NEDD4 and other HECT E3 ligase subfamilies, in GC.

ETS1 is a prognostic biomarker of triple-negative breast cancer and promotes the triple-negative breast cancer progression through the YAP signaling.

E26 transcription factor-1 (ETS1) is involved in extracellular matrix remodeling, migratory infiltration and angiogenesis in tumors and known to play an important role in tumor progression. However, the mechanism by which ETS1 promotes tumor progression remains elusive. In this report, we show that ETS1 is highly expressed in breast tumor tissues and specifically associated with the tumor metastasis and poor survival in triple negative breast cancer (TNBC) tumors, upon analysis by immunohistochemical (IHC) staining of tumor samples from 240 breast cancer cases. Depletion of ETS1 in TNBC cells by shETS1 significantly inhibited the cell proliferation and migration. Mechanistically, knockdown of ETS1 in TNBC cells dramatically reduced expression of YAP and the YAP target genes, and overexpression of YAP in the ETS1 knockdown cells restored the cell proliferation and migration. These data indicate that YAP is a downstream effector mediating the ETS1-promoted TNBC cell proliferation and migration. Taken together, our results suggest that ETS1 promotes TNBC progression through the YAP signaling.

Overexpression of SCYL1 Is Associated with Progression of Breast Cancer.

SCYL1 is a pseudokinase and plays roles in cell division and gene transcription, nuclear/cytoplasmic shuttling of tRNA, protein glycosylation, and Golgi morphology. However, the role of SCYL1 in human breast cancer progression remains largely unknown. In this study, we determined expression of SCYL1 in breast cancer by searching the Cancer Genome Atlas (TCGA) and Tumor Immunoassay Resource (TIMER) databases. Meanwhile, we collected breast tumor tissue samples from 247 cases and detected expression of SCYL1 in the tumors using the tissue microarray assay (TMA). Association of SCYL1 with prognosis of breast cancer was determined based on the PrognoScan database. The results have shown that SCYL1 is overexpressed in breast cancer, and the expression of SCYL1 is associated with poor clinical outcomes of breast cancer patients. Furthermore, knockdown of SCYL1 by shRNAs significantly inhibited the proliferation and migration of breast cancer cells. Taken together, our data suggest that SCYL1 is a biomarker for poor prognosis of breast cancer, has a promoting role in breast cancer progression, and is a potential target for breast cancer therapy.

Geranylgeranylation signaling promotes breast cancer cell mitosis via the YAP-activated transcription of kinetochore/centromere genes.

Geranylgeranylation signaling plays an important role in cancer cell proliferation. Our previous studies have shown that the YAP is one of the geranylgeranylation signal transducers in breast cancer cells (Mi W, et al., Oncogene. 2015; 34(24): 3095-3106). However, the downstream effectors that mediate the promoting effect of the geranylgeranylation/YAP signal axis on breast cancer cell proliferation remain elusive. In this report, we investigated the pathway that mediates the effect of the geranylgeranylation on breast cancer cell proliferation. The results have shown that inhibition of geranylgeranyl biosynthesis inactivates transcription of a set of kinetochore/centromere genes. Further biochemical and cell biological studies demonstrated that inhibition of geranylgeranyl biosynthesis significantly reduced the level of key kinetochore/centromere proteins, thus caused a defect in mitosis. Knockdown of YAP caused similar inhibitory effects on the kinetochore/centromere gene expression and mitosis to that of inhibition of geranylgeranyl biosynthesis. Furthermore, we found that E2F1, the gene coding for E2F1 that is known to activate expression of cell cycle genes, is a target gene of YAP. Knockdown of E2F1 also reduced expression of the kinetochore/centromere genes, suggesting that the activation effect of YAP on expression of the kinetochore/centromere genes may be mediated by E2F1. Our studies have proposed a novel geranylgeranylation-dependent cancer cell proliferation signaling pathway in which geranylgeranylation signaling promotes cancer cell mitosis via the YAP-activated transcription of kinetochore/centromere genes.

Potential roles for pattern molecule of PAMP-triggered immunity in improving crop cold tolerance.

KEY MESSAGE: The application of flagellin 22 (flg22), the most widely studied PAMP, enhance crop cold tolerance. ICE1-CBF pathway and SA signaling is involved in the alleviation of cold injury by flg22 treatment. Pathogen infection cross-activates cold response and increase cold tolerance of host plants. However, it is not possible to use the infection to increase cold tolerance of field plants. Here flagellin 22 (flg22), the most widely studied PAMP (pathogen-associated molecular patterns), was used to mimic the pathogen infection to cross-activate cold response. Flg22 treatment alleviated the injury caused by freezing in Arabidopsis, oilseed and tobacco. In Arabidopsis, flg22 activated the expression of immunity and cold-related genes. Moreover, the flg22 induced alleviation of cold injury was lost in NahG transgenic line (SA-deficient), sid2-2 and npr1-1 mutant plants, and flg22-induced expression of cold tolerance-related genes, which indicating that salicylic acid signaling pathway is required for the alleviation of cold injury by flg22 treatment. In short flg22 application can be used to enhance cold tolerance in field via a salicylic acid-depended pathway.

The Role of CXC Chemokines in Cancer Progression.

CXC chemokines are small chemotactic and secreted cytokines. Studies have shown that CXC chemokines are dysregulated in multiple types of cancer and are closely correlated with tumor progression. The CXC chemokine family has a dual function in tumor development, either tumor-promoting or tumor-suppressive depending on the context of cellular signaling. Recent evidence highlights the pro-tumorigenic properties of CXC chemokines in most human cancers. CXC chemokines were found to play pivotal roles in promoting angiogenesis, stimulating inflammatory responses, and facilitating tumor metastases. Enhanced expression of CXC chemokines is always signatured with inferior survival and prognosis. The levels of CXC chemokines in cancer patients are in dynamic change according to the tumor contexts (e.g., chemotherapy resistance and tumor recurrence after surgery). Thus, CXC chemokines have great potential to be used as diagnostic and prognostic biomarkers and therapeutic targets. Currently, the molecular mechanisms underlying the effect of CXC chemokines on tumor inflammation and metastasis remain unclear and application of antagonists and neutralizing antibodies of CXC chemokines signaling for cancer therapy is still not fully established. This article will review the roles of CXC chemokines in promoting tumorigenesis and progression and address the future research directions of CXC chemokines for cancer treatment.

MEKK5 Interacts with and Negatively Regulates the E3 Ubiquitin Ligase NEDD4 for Mediating Lung Cancer Cell Migration.

Our previous studies have shown that the HECT E3 ubiquitin ligase NEDD4 and kinase MEKK5 both play an essential role in lung cancer migration. A report predicts that MEKK5 may be ubiquitinated by NEDD4; however, interaction of MEKK5 with NEDD4 and ubiquitination of MEKK5 by NEDD4 have not been characterized. In this report, we show that NEDD4 interacts with MEKK5 through a conserved WW3 domain by the co-immunoprecipitation and the GST-pulldown assays. The ubiquitination assay indicates that MEKK5 is not a ubiquitination substrate of NEDD4, but negatively regulates NEDD4-mediated ubiquitination. Furthermore, overexpression of MEKK5 significantly reduced the NEDD4-promoted lung cancer cell migration. Taken together, our studies have defined an inhibitory role of MEKK5 in regulation of NEDD4-mediated ubiquitination.

Restoration of the ATG5-dependent autophagy sensitizes DU145 prostate cancer cells to chemotherapeutic drugs.

Autophagy serves an important role in cancer cell survival and drug resistance. In the present study, the prostate cancer DU145 cell line was used, which lacks autophagy related 5 (ATG5) expression and is defective in induction of ATG5-dependent autophagy. The aim of the study was to examine the effects of the restoration of autophagy on cell proliferation and migration, and to assess the cytotoxicity caused by chemotherapeutic drugs, using microscopic, wound-healing, western blot and apoptotic assays. The restoration of the autophagic activity in DU145 cells by the overexpression of ATG5 enhanced the cell proliferation and migration rates. Notably, restoration of the ATG5-dependent autophagy in DU145 cells significantly increased the cytotoxic effects of the chemotherapeutic drugs, docetaxel and valproic acid, and the endoplasmic reticulum stress inducers, brefeldin A, tunicamycin and thapsigargin. The present study provides a novel perspective on the role of ATG5-dependent autophagy in drug resistance and chemotherapy.

Geranylgeranylation promotes proliferation, migration and invasion of gastric cancer cells through the YAP signaling pathway.

Geranylgeranylation (GGylation) is a lipid modification process of signaling proteins. Currently, very little is known about the GGylation signaling for gastric cancer cell proliferation and migration. In this report, we found that inhibition of GGylation by the mevalonate pathway inhibitor atorvastatin and the geranylgeranyltransferase I inhibitor GGTI-298 impairs proliferation and migration of the gastric cancer AGS cells. During searching the signaling pathway for the effect, we observed that YAP, a transcription activator and downstream effector of the hippo pathway, was suppressed by inhibition of GGylation, as evaluated by detection of the mRNA level of its known target genes CYR61 and CTGF and translocation to nuclei. Knockdown of YAP by shRNAs produced a similar effect on proliferation and migration of gastric cancer AGS cells to that of GGylation inhibition, suggesting that GGylation signaling promotes gastric cancer cell proliferation and migration by activation of YAP. Our studies provide a potential new therapeutic targeting pathway for gastric cancer.

ASK1 inhibits proliferation and migration of lung cancer cells via inactivating TAZ.

ASK1 (Apoptosis Signal-regulating Kinase 1, also MEKK5) is known to mediate cellular stress signaling pathways through activating p38 kinase. We here observed that ectopically expression of ASK1, but not its kinase-dead mutant, impaired cell proliferation and migration in lung cancer A549 and NCI-H1975 cells. To our surprise, this inhibitory effect of ASK1 is independent on activation of p38 kinase. We further discovered that ASK1 interacts with the WW domain of YAP and TAZ (also WWTR1) that are transcriptional co-activators and the Hippo signaling effectors. Overexpression of wild type ASK1, but not the kinase-dead mutant, in the lung cancer cells down-regulated the expression of the YAP/TAZ target genes CYR61 and CTGF. It seems that ASK1 specifically inactivates TAZ, not YAP, as ASK1 blocked nuclear translocation of TAZ only, while had no effect on YAP. Furthermore, knockdown of TAZ in the lung cancer cells caused the same inhibitory effect on cell proliferation and migration as that of overexpression of ASK1. Thus, our studies have defined a new signaling pathway of ASK1 for regulation of lung cancer cell proliferation and migration via interacting with and inactivating TAZ.

The Hippo signaling effector WWTR1 is a metastatic biomarker of gastric cardia adenocarcinoma.

BACKGROUND: Gastric cardia adenocarcinoma (GCA) is an aggressive subtype of gastric cancer with a high metastatic rate. However, the metastatic biomarker of GCA has not been established. METHODS: To search for the biomarker for GCA metastasis, we here examined expression of the Hippo signaling effector WWTR1 (WW domain containing transcription regulator 1, commonly listed as TAZ) in tumor tissue samples from 214 GCA cases using the tissue microarray assay (TMA), and statistically analyzed association of the WWTR1 expression with metastasis-related pathological outcomes and cumulative survival of the GCA patients. Furthermore, shRNA knockdown was used to determine the role of WWTR1 in promoting cell migration in gastric cancer cells. RESULTS: The results have shown that WWTR1 is overexpressed in 66.4% of the GCA tumor samples. Expression of WWTR1 has a significant inverse correlation with cumulative survival of GCA patients (p < 0.01). WWTR1 positive patients had a mean survival of 56.9 ± 4.4 months, comparing to WWTR1 negative mean survival of 77.3 ± 5.9 months. More importantly, expression of WWTR1 significantly associated with tumor invasion and metastasis (in T stage, p = 0.031; N stage, p < 0.01; and TNM stage, p < 0.001). Furthermore, knockdown of WWTR1 impaired migration of gastric cancer AGS cells. CONCLUSIONS: Our studies have identified WWTR1 as a metastatic biomarker of GCA for poor prognosis, defined a role of WWTR1 in driving metastasis of gastric cancer, and suggested WWTR1 as a potential target for anti-metastatic therapy of GCA.

Regulation of Krüppel-like factor 8 by the NEDD4 E3 ubiquitin ligase.

Krüppel-like factor 8 (KLF8) plays many important roles in various diseases, especially cancer. Previous studies have shown that KLF8 is regulated by ubiquitylation. The molecular mechanism underlying this posttranslational modification of KLF8, however, has not been investigated. Reported here is our identification of the neural precursor cell expressed, developmentally down-regulated 4 (NEDD4) as the E3 ubiquitin ligase for this modification. By co-immunoprecipitation and ubiquitylation assays, we determined that KLF8 interacts with NEDD4 and is ubiquitylated by NEDD4. By site-directed mutagenesis and pharmacological inhibition of MEK, we found that the ubiquitylation of KLF8 by NEDD4 depends upon the phosphorylation of KLF8 at serine 48 by ERK. Cycloheximide chase analysis, target gene promoter reporter assay and fluorescent staining indicated that NEDD4 plays a critical role in promoting the stability and transcriptional activity of KLF8 in the nucleus. Taken together, this work identified NEDD4 as a novel E3 ubiquitin ligase for KLF8 that provides insights into targeting the KLF8-NEDD4 axis to treat various types of cancer associated with overexpression of both proteins.

The E3 ubiquitin ligase NEDD4 mediates cell migration signaling of EGFR in lung cancer cells.

BACKGROUND: EGFR-dependent cell migration plays an important role in lung cancer progression. Our previous study observed that the HECT E3 ubiquitin ligase NEDD4 is significantly correlated with tumor metastasis and required for migration and invasion signaling of EGFR in gastric cancer cells. However, how NEDD4 promotes the EGFR-dependent lung cancer cell migration is unknown. This study is to elucidate the mechanism by which NEDD4 mediates the EGFR lung cancer migration signaling. METHODS: Lentiviral vector-loaded NEDD4 shRNA was used to deplete endogenous NEDD4 in lung cancer cell lines. Effects of the NEDD4 knockdown on the EGFR-dependent or independent lung cancer cell migration were determined using the wound-healing and transwell assays. Association of NEDD4 with activated EGFR was assayed by co-immunoprecipitation. Co-expression of NEDD4 with EGFR or PTEN was determined by immunohistochemical (IHC) staining in 63 lung adenocarcinoma tissue samples. Effects of NEDD4 ectopic expression or knockdown on PTEN ubiquitination and down-regulation, AKT activation and lysosomal secretion were examined using the GST-Uba pulldown assay, immunoblotting, immunofluorescent staining and a human cathepsin B ELISA assay respectively. The specific cathepsin B inhibitor CA-074Me was used for assessing the role of cathepsin B in lung cancer cell migration. RESULTS: Knockdown of NEDD4 significantly reduced EGF-stimulated cell migration in non-small cell lung carcinoma (NSCLC) cells. Co-immunoprecipitation assay found that NEDD4 is associated with EGFR complex upon EGF stimulation, and IHC staining indicates that NEDD4 is co-expressed with EGFR in lung adenocarcinoma tumor tissues, suggesting that NEDD4 might mediate lung cancer cell migration by interaction with the EGFR signaling complex. Interestingly, NEDD4 promotes the EGF-induced cathepsin B secretion, possibly through lysosomal exocytosis, as overexpression of the ligase-dead mutant of NEDD4 impedes lysosomal secretion, and knockdown of NEDD4 significantly reduced extracellular amount of cathepsin B induced by EGF. Consistent with the role of NEDD4, cathepsin B is pivotal for both basal and the EGF-stimulated lung cancer cell migration. Our studies propose a novel mechanism underlying the EGFR-promoted lung cancer cell migration that is mediated by NEDD4 through regulation of cathepsin B secretion. CONCLUSION: NEDD4 mediates the EGFR lung cancer cell migration signaling through promoting lysosomal secretion of cathepsin B.

The E3 ubiquitin ligase NEDD4 is an LC3-interactive protein and regulates autophagy.

The MAP1LC3/LC3 family plays an essential role in autophagosomal biogenesis and transport. In this report, we show that the HECT family E3 ubiquitin ligase NEDD4 interacts with LC3 and is involved in autophagosomal biogenesis. NEDD4 binds to LC3 through a conserved WXXL LC3-binding motif in a region between the C2 and the WW2 domains. Knockdown of NEDD4 impaired starvation- or rapamycin-induced activation of autophagy and autophagosomal biogenesis and caused aggregates of the LC3 puncta colocalized with endoplasmic reticulum membrane markers. Electron microscopy observed gigantic deformed mitochondria in NEDD4 knockdown cells, suggesting that NEDD4 might function in mitophagy. Furthermore, SQSTM1 is ubiquitinated by NEDD4 while LC3 functions as an activator of NEDD4 ligase activity. Taken together, our studies define an important role of NEDD4 in regulation of autophagy.

The Hippo-YAP/TAZ pathway mediates geranylgeranylation signaling in breast cancer progression.

Protein geranylgeranylation (GGylation) regulates the function of various signal transducers including small GTPases and Ggamma subunits. The role of GGylation in breast cancer progression is poorly understood. Recent studies suggest that GGylation promotes the proliferation and migration of breast carcinoma cells through the Hippo-YAP/TAZ pathway.

Stress-induced endocytosis and degradation of epidermal growth factor receptor are two independent processes.

BACKGROUND: Epidermal growth factor receptor (EGFR) is an important oncogenic protein in multiple types of cancer. Endocytosis and degradation of epidermal growth factor receptor (EGFR) are two key steps for down-regulation of cell surface level of EGFR and modulation of EGFR signaling. Stress conditions induce ligand-independent endocytosis and degradation of EGFR. However, it is not clear whether stress-induced endocytosis and degradation are consequential or two independent events. METHODS: Endocytosis and degradation of EGFR in response to stress treatment and effects of the p38 inhibitor, the Caspase-3 inhibitor and the proteasomal inhibitor in cervical cancer HeLa cells were determined using immunoblotting and immunofluorescent staining assays. RESULTS: Stress conditions, such as protein biosynthesis inhibition, UV light irradiation, and hyper-osmosis, induced both ligand-independent endocytosis and degradation of EGFR. Stress-induced endocytosis of EGFR relies on p38 kinase activity, while stress-induced degradation of EGFR is catalyzed by Caspase-3 activity. Inhibiting p38 kinase impairs only the endocytosis but not the degradation, while inhibiting Caspase-3 results in the opposite effect to inhibiting p38. Furthermore, proteasomal activity is required for stress-induced degradation of EGFR and cell death, but not for endocytosis. CONCLUSIONS: The results indicate that stress-induced endocytosis and degradation are two independent events and suggest stress signaling may utilize a double-secure mechanism to down-regulate cell surface EGFR in cancer cells.

CYR61 (CCN1) is a metastatic biomarker of gastric cardia adenocarcinoma.

Gastric cardia adenocarcinoma (GCA) is the most aggressive subtype of gastric cancer with a high metastatic rate. In this report, we collected tumor tissue samples from 214 GCA cases and examined expression of CYR61, a target gene product of the Hippo-YAP/TAZ pathway, in the GCA tumors by immunohistochemical (IHC) staining using the tissue microarray assay (TMA). The results have shown that CYR61 is overexpressed in 44% of the GCA tumor samples. Expression of CYR61 is inversely correlated with cumulative survival of GCA patients (p<0.001) and significantly associated only with metastatic pathological categories (with N category, p=0.052; with TNM stage, p=0.001). Furthermore, knockdown of CYR61 in gastric cancer AGS cells impairs the cancer cell migration and invasion, suggesting a driver role of CYR61 in metastasis. Thus, our studies have established CYR61 as a metastatic biomarker for prediction of poor prognosis of GCA and provided a potential molecular target for anti-metastatic therapy of GCA.

PBK/TOPK mediates geranylgeranylation signaling for breast cancer cell proliferation.

PDZ binding-kinase (PBK) (also named T-lymphokine-activated killer cell-originated protein kinase (TOPK)), a serine/threonine kinase, is tightly controlled in normal tissues but elevated in many tumors, and functions in tumorigenesis and metastasis. However, the signaling that regulates expression of PBK in cancer cells remains elusive. Here we show that atorvastatin (Lipitor), an inhibitor of hydroxymethylglutaryl co-enzyme A (HMG-CoA) reductase that is a rate-limiting enzyme of mevalonate pathway, down-regulates expression of PBK by impairing protein geranylgeranylation. The shRNA knockdown demonstrated that Yes-associated protein (YAP) mediates geranylgeranylation-regulated expression of PBK. Importantly, atorvastatin or the geranylgeranyltransferase I inhibitor GGTI-298 inhibited breast cancer cell proliferation through inactivation of YAP signaling and down-regulation of PBK. These findings have defined a new signaling pathway that regulated expression of PBK and identified PBK as a downstream target of the Hippo-YAP signaling, uncoverd a mechanism underlying the anti-cancer effect by inhibition of mevalonate pathway and geranylgeranylation, and provided a potential target for breast cancer targeted therapy.