WBP2 promotes gastric cancer cell migration via novel targeting of LATS2 kinase in the Hippo tumor suppressor pathway.

Dysregulation of signaling pathways is responsible for many human diseases. The lack of understanding of the molecular etiology of gastric cancer (GC) poses a substantial challenge to the development of effective cancer therapy. To better understand the molecular mechanisms underlying the pathogenesis of GC, which will facilitate the identification and development of effective therapeutic approaches to improve patient outcomes, mass spectrometry-based phosphoproteomics analysis was performed to map the global molecular changes in GC. A total of 530 proteins with altered phosphorylation levels were detected across a panel of 15 normal and GC cell lines. WW domain-binding protein 2 (WBP2) was validated to be upregulated in a subset of GC cell lines. WBP2 is overexpressed in 61% cases of GC compared to non-cancer tissues and high WBP2 expression correlates with poor clinical outcomes. WBP2 was found to be required for GC cell migration but is dispensable for cell growth and proliferation. WBP2 knockdown increased p-LATS2 with a concomitant increase in p-YAP, resulting in the cytoplasmic retention of YAP and ultimately the inhibition of YAP/TEAD activity and downregulation of TEAD target genes--CTGF and CYR61. Importantly, the loss of LATS2 reversed the activation of Hippo pathway caused by WBP2 knockdown, indicating that WBP2 acts through LATS2 to exert its function on the Hippo pathway. Moreover, WBP2 interacted with LATS2 to inhibit its phosphorylation and activity. In conclusion, our study established a pivotal role for WBP2 in the promotion of GC cell migration via a novel mechanism that inactivates the Hippo pathway transducer LATS2.

Comparative analysis of the catalytic regulation of NEDD4-1 and WWP2 ubiquitin ligases.

NEDD4-1 E3 ubiquitin protein ligase (NEDD4-1) and WW domain-containing E3 ubiquitin ligase (WWP2) are HECT family ubiquitin E3 ligases. They catalyze Lys ubiquitination of themselves and other proteins and are important in cell growth and differentiation. Regulation of NEDD4-1 and WWP2 catalytic activities is important for controlling cellular protein homeostasis, and their dysregulation may lead to cancer and other diseases. Previous work has implicated noncatalytic regions, including the C2 domain and/or WW domain linkers in NEDD4-1 and WWP2, in contributing to autoinhibition of the catalytic HECT domains by intramolecular interactions. Here, we explored the molecular mechanisms of these NEDD4-1 and WWP2 regulatory regions and their interplay with allosteric binding proteins such as Nedd4 family-interacting protein (NDFIP1), engineered ubiquitin variants, and linker phosphomimics. We found that in addition to influencing catalytic activities, the WW domain linker regions in NEDD4-1 and WWP2 can impact product distribution, including the degree of polyubiquitination and Lys-48 versus Lys-63 linkages. We show that allosteric activation by NDFIP1 or engineered ubiquitin variants is largely mediated by relief of WW domain linker autoinhibition. WWP2-mediated ubiquitination of WW domain-binding protein 2 (WBP2), phosphatase and tensin homolog (PTEN), and p62 proteins by WWP2 suggests that substrate ubiquitination can also be influenced by WW linker autoinhibition, although to differing extents. Overall, our results provide a deeper understanding of the intricate and multifaceted set of regulatory mechanisms in the control of NEDD4-1-related ubiquitin ligases.

WBP2 Downregulation Inhibits Proliferation by Blocking YAP Transcription and the EGFR/PI3K/Akt Signaling Pathway in Triple Negative Breast Cancer.

BACKGROUND/AIMS: Dysregulated expression of WW domain-binding protein 2 (WBP2) is associated with poor prognosis in ER+ breast cancer patients. However, its role in triple negative breast cancer (TNBC) has not been previously assessed. Therefore, we aimed to elucidate the functional mechanism of WBP2 in TNBC cells. METHODS: qRT-PCR, western blotting, and immunohistochemical staining were used to evaluate WBP2 expression in TNBC patient tumors and cell lines. HCC1937 and MDA-MB-231 cells transiently transfected with WBP2 small interfering RNA (siRNA), miR-613 mimics, or miR-613 inhibitors were subject to assays for cell viability, apoptosis and cell cycle distribution. Co-immunoprecipitation, western blotting or qRT-PCR were employed to monitor changes in signaling pathway-related genes and proteins. Luciferase assays were performed to assess whether WBP2 is a direct target of miR-613. The effect of miR-613 on tumor growth was assessed in vivo using mouse xenograft models. RESULTS: The expression of WBP2 was upregulated in TNBC tissues and cells. Expression of WBP2 was significantly correlated with Ki67 in TNBC patients. Knockdown of WBP2 inhibited cellular proliferation, promoted apoptosis, and induced cell cycle arrest of TNBC cells. miR-613 directly bound to the 3'-untranslated region (3'-UTR) of WBP2 and regulated the expression of WBP2. Moreover, miR-613 reduced the expression of WBP2 and suppressed tumor growth of TNBC cells in vivo. Knockdown of WBP2 inhibited YAP transcription and the EGFR/PI3K/Akt signaling pathway in TNBC cells, and these effects were reversed by inhibition of miR-613. CONCLUSION: WBP2 overexpression is associated with the poor prognosis of TNBC patients and the miR-613-WBP2 axis represses TNBC cell growth by inactivating YAP-mediated gene expression and the EGFR/PI3K/Akt signaling pathway.

Is there an association between PAWP/WBP2NL sequence, expression, and distribution in sperm cells and fertilization failures in ICSI cycles?

Successful fertilization in mammals depends on the sperm's ability to initiate intracellular Ca2+ oscillations in the egg, a phenomenon that is elicited by Sperm-oocyte activating factors (SOAFs), whose quantitative and/or qualitative defect might result in fertilization failure. One such proposed factor is Post-acrosomal WW domain-binding protein (PAWP/WBP2NL), although its ability to activate human oocytes has been questioned and its implication in human fertilization failure remains unknown. Here, we sought to determine if PAWP/WBP2NL expression and distribution in sperm cells associate with low/complete fertilization failure in males participating in intracytoplasmic sperm injection (ICSI) cycles. This prospective study was conducted on eight couples referred for elective ICSI with either the woman's own (n = 4) or a donor eggs (n = 4). Eight sperm donor samples used in ICSI, which resulted in normal fertilization rates, were used as the control group. For each male patient and donor sperm, PAWP/WBP2NL sequence, protein expression, and cellular distribution were analyzed by PCR amplification-sequencing, Western blot, and immunofluorescence, respectively. PAWP/WBP2NL was present in all samples, and no significant differences were detected between patients with fertilization failure and donors in sequence variants or mean protein expression, or in the proportion of PAWP/WBP2NL-positive sperm. In conclusion, no clear association between PAWP/WBP2NL protein expression in sperm and fertilization outcome in ICSI were observed from this cohort.

WW domain-binding protein 2: an adaptor protein closely linked to the development of breast cancer.

The WW domain is composed of 38 to 40 semi-conserved amino acids shared with structural, regulatory, and signaling proteins. WW domain-binding protein 2 (WBP2), as a binding partner of WW domain protein, interacts with several WW-domain-containing proteins, such as Yes kinase-associated protein (Yap), paired box gene 8 (Pax8), WW-domain-containing transcription regulator protein 1 (TAZ), and WW-domain-containing oxidoreductase (WWOX) through its PPxY motifs within C-terminal region, and further triggers the downstream signaling pathway in vitro and in vivo. Studies have confirmed that phosphorylated form of WBP2 can move into nuclei and activate the transcription of estrogen receptor (ER) and progesterone receptor (PR), whose expression were the indicators of breast cancer development, indicating that WBP2 may participate in the progression of breast cancer. Both overexpression of WBP2 and activation of tyrosine phosphorylation upregulate the signal cascades in the cross-regulation of the Wnt and ER signaling pathways in breast cancer. Following the binding of WBP2 to the WW domain region of TAZ which can accelerate migration, invasion and is required for the transformed phenotypes of breast cancer cells, the transformation of epithelial to mesenchymal of MCF10A is activated, suggesting that WBP2 is a key player in regulating cell migration. When WBP2 binds with WWOX, a tumor suppressor, ER transactivation and tumor growth can be suppressed. Thus, WBP2 may serve as a molecular on/off switch that controls the crosstalk between E2, WWOX, Wnt, TAZ, and other oncogenic signaling pathways. This review interprets the relationship between WBP2 and breast cancer, and provides comprehensive views about the function of WBP2 in the regulation of the pathogenesis of breast cancer and endocrine therapy in breast cancer treatment.

WBP2NL/PAWP mRNA and protein expression in sperm cells are not related to semen parameters, fertilization rate, or reproductive outcome.

PURPOSE: WBP2NL/PAWP, a protein found in the post-acrosomal region of mammalian spermatozoa, has been proposed as a sperm-borne oocyte-activating factor (SOAF) contributing to Ca2+ release within the oocyte and subsequent fertilization and embryo development. However, its relevance as either a diagnostic or a prognostic marker of fertilization failure has been questioned in the recent literature. We analyzed WBP2NL/PAWP gene and protein expression level and localization in patients without previous intracytoplasmic sperm injection (ICSI) cycles in order to assess its association with both sperm characteristics and ability to fertilize. METHODS: Raw frozen-thawed semen samples from 33 couples referred for oocyte donation were included in the study during 2015. Relative protein expression versus α-tubulin (western blot, WB), proportion of post-acrosomal WBP2NL/PAWP-positive spermatozoa over the total number of sperm cells (immunofluorescence), and WBP2NL/PAWP gene expression (RT-qPCR) were analyzed and correlated with semen analysis parameters (number, motility, and morphology) and with reproductive outcomes. RESULTS: WBP2NL/PAWP protein was expressed in all samples with high variability: relative protein expression (1.77 ± 0.8, range [0.4-3.7]), proportion of positive cells (49.6% ± 16.1, range [22-89]), and relative gene expression (7.3 ± 8.2). No significant correlation (R 2 < 0.1) was found between gene and protein expression, neither between WBP2NL/PAWP gene or protein expression, and fertilization rate or other reproductive outcomes (i.e., pregnancy). In contrast, we found significant correlation between sperm morphology and WBP2NL/PAWP semiquantitative analysis in WB (r = -0.42, p < 0.05) and for sperm motility and WBP2NL/PAWP expression in IF (r = 0.52, p < 0.05). CONCLUSION: Taken into account that WBP2NL/PAWP gene and protein levels and distribution did not correlate with fertilization rates, this study questions the interest of WBP2NL/PAWP protein and gene expression analysis in sperm cells as a prognostic factor for the outcome of ICSI cycles. Larger studies focusing on WBP2NL/PAWP protein and gene expression are needed in order to evaluate the role of WBP2NL/PAWP as a prognostic factor for ART.

Proteomic detection of COX-2 pathway-related factors in patients with adenomyosis.

Background: Investigating the relationship between cyclooxygenase-2 (COX-2) pathway-related factors and clinical features in patients with adenomyosis by proteomics could provide potential therapeutic targets. Methods: This study recruited 40 patients undergoing surgical hysterectomy and pathological diagnosis of adenomyosis, collected ectopic endometrial specimens, and recorded clinical data. The expression levels of COX-2 in ectopic uterus lesions were detected using the immunohistochemical (IHC) SP method. The 40 samples were then divided into a COX-2 low or high expression group. Five samples with the most typical expression levels were selected from each of the two groups and the differential proteins between the two groups were identified using label-free quantitative proteomics. WW domain-binding protein 2 (WBP2), interferon induced transmembrane protein 3 (IFITM3), and secreted frizzled-related protein 4 (SFRP4) were selected for further verification, and their relationships with COX-2 and clinical characteristics were analyzed. Results: There were statistically significant differences in the expression of WBP2, IFITM3, and SFRP4 between the COX-2 low and high expression groups (P < 0.01). The expressions of COX-2, IFITM3, and SFRP4 were significantly correlated with dysmenorrhea between the two groups (P < 0.05), but not with uterine size or menstrual volume (P > 0.05). However, there was no significant correlation between the expression of WBP2 and dysmenorrhea, uterine size, and menstruation volume in both the high expression and low expression groups (P > 0.05). Conclusions: COX-2, IFITM3, SFRP4, and WBP2 may be involved in the pathogenesis of adenomyosis. COX-2, IFITM3, and SFRP4 may serve as potential molecular biomarkers or therapeutic targets in dysmenorrhea in patients with early adenomyosis.

WBP2 restrains the lysosomal degradation of GPX4 to inhibit ferroptosis in cisplatin-induced acute kidney injury.

Cisplatin is one of the major causes of acute kidney injury (AKI) in clinical practice, and ferroptosis is an essential form of cell death in cisplatin-induced AKI (CP-AKI). WW domain binding protein-2 (WBP2), a molecular chaperon, is involved in the progression of various malignancies, but its role in renal injuries has not been investigated. Our present study employed bioinformatics analysis to identify WBP2 as a potential modulator of AKI and ferroptosis. Preliminary laboratory investigations showed that WBP2, highly expressed in renal proximal tubular cells, was downregulated in CP-AKI. Further studies demonstrated that WBP2 decelerated ferroptosis to alleviate CP-AKI. Mechanistically, WBP2 interacted with glutathione peroxidase 4 (GPX4, a key detoxicating enzyme for ferroptosis) via its PPXY1 motif to inhibit ferroptosis. Furthermore, the in-depth investigations revealed that WBP2 competed with heat shock cognate protein 70 (HSC70) for the binding with the KEFRQ-like motifs of GPX4, leading to the deceleration of chaperon-mediated autophagy of GPX4. All in all, this study indicated the beneficial effect of WBP2 in CP-AKI and its relevance with ferroptosis, thus providing a novel insight into the modulation of ferroptosis in cisplatin-related nephropathy.

WBP2 promotes BTRC mRNA stability to drive migration and invasion in triple-negative breast cancer via NF-κB activation.

WW-domain-binding protein 2 (WBP2) is an oncogene that drives breast carcinogenesis through regulating Wnt, estrogen receptor (ER), and Hippo signaling. Recent studies have identified neoteric modes of action of WBP2 other than its widely recognized function as a transcriptional coactivator. Here, we identified a previously unexplored role of WBP2 in inflammatory signaling in breast cancer via an integrated proteogenomic analysis of The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA BRCA) dataset. WBP2 was shown to enhance the migration and invasion in triple-negative breast cancer (TNBC) cells especially under tumor necrosis factor alpha (TNF-α) stimulation. Molecularly, WBP2 potentiates TNF-α-induced nuclear factor kappa B (NF-κB) transcriptional activity and nuclear localization through aggrandizing ubiquitin-mediated proteasomal degradation of its upstream inhibitor, NF-κB inhibitor alpha (NFKBIA; also known as IκBα). We further demonstrate that WBP2 induces mRNA stability of beta-transducin repeat-containing E3 ubiquitin protein ligase (BTRC), which targets IκBα for ubiquitination and degradation. Disruption of IκBα rescued the impaired migratory and invasive phenotypes in WBP2-silenced cells, while loss of BTRC ameliorated WBP2-driven migration and invasion. Clinically, the WBP2-BTRC-IκBα signaling axis correlates with poorer prognosis in breast cancer patients. Our findings reveal a pivotal mechanism of WBP2 in modulating BTRC-IκBα-NF-κB pathway to promote TNBC aggressiveness.

WW domain binding protein 2 (WBP2) as an oncogene in breast cancer: mechanisms and therapeutic prospects-a narrative review.

Background and Objective: WW domain binding protein 2 (WBP2), considered an emerging breast cancer gene, functions as a binding partner for WW domain proteins. The WBP2 gene is involved in mediating the malignant development and clinical drug resistance of breast cancer, but its potential mechanism remains unclear. Therefore, it is necessary to elucidate the mechanism of WBP2 in breast cancer, which will help to provide new methods for clinical diagnosis and treatment of breast cancer. Methods: The PubMed database was searched using the terms "WW Domain Binding Protein 2" or "WBP2", "breast cancer" or "breast neoplasms" or "human cancer" from January 1997 through August 2022. Through the screening and evaluation of titles and abstracts, about 120 English articles were included in this study. Key Content and Findings: By describing the multiple regulatory functions of WBP2 at the transcriptional, post-transcriptional, and post-translational levels, and summarizing how WBP2 as a key node crosstalks multiple signaling pathways, we reveal the ability of WBP2 to promote breast cancer malignant progression. In different subtypes of breast cancer, the mechanism of WBP2-mediated drug resistance is related to estrogen receptor and epidermal growth factor receptor (EGFR) 2 status, and hormones may be an essential factor in WBP2-mediated drug resistance. In addition, we discuss the application prospects of WBP2 in targeted therapy and immunotherapy and propose therapeutic strategies to overcome drug resistance in breast cancer by jointly targeting WBP2 and its related molecules. This provides a theoretical basis for the innovation of breast cancer targeted drugs. Conclusions: WBP2 is a promising target for breast cancer therapy. Nuclear WBP2, as the main functional form of WBP2 after its activation, is a meaningful indicator for the diagnosis and prediction of breast cancer progression.

WBP2 modulates G1/S transition in ER+ breast cancer cells and is a direct target of miR-206.

PURPOSE: The mechanisms underlying the oncogenic properties of WW domain binding protein 2 (WBP2) in breast cancer have not been fully understood. In this study, we explored the role of WBP2 in cell cycle regulation in ER+ breast cancer cells and how it is regulated in the cancer cells. METHODS: The association between WBP2 expression and prognosis in ER+ breast cancer was assessed by data mining in Breast Cancer Gene-Expression Miner v4.0. Cell cycle was assessed by PI staining and flow cytometry. EdU staining was applied to visualize cells in S phase. The binding between miR-206 and WBP2 were verified by dual luciferase assay. CCK-8 assay and flow cytometric analysis were applied to assess the functional role of WBP2 and miR-206 in the cancer cells. RESULTS: High WBP2 expression correlates with higher risk of any events (AE) and metastatic relapse (MR) and also indicates shorter AE-free survival and MR-free survival in ER+ breast cancer patients. In both MCF-7 and BT474 cells, WBP can influence the expression of G1/S-related cell cycle proteins, including p21, CDK4, and cyclin D1. In addition, WBP2 overexpression resulted in facilitated G1/S transition, while WBP2 knockdown impaired the transition. The 3'UTR of WBP2 has a conserved miR-206 binding site. Functionally, miR-206 knockdown decreased tamoxifen sensitivity in tamoxifen-sensitive (TamS) MCF-7 cells, while miR-206 overexpression and WBP2 knockdown enhanced the sensitivity in tamoxifen-resistant (TamR) MCF-7 cells. CONCLUSION: Based on these findings, we infer that the miR-206/WBP2 axis can modulate tamoxifen sensitivity via regulating G1/S progression in ER+ breast cancer.

Elevated Expression of the Testis-specific Gene WBP2NL in Breast Cancer.

Breast cancer is one of the most common causes of cancer death in women; therefore, the study of molecular aspects of breast cancer for finding new biomarkers is important. Recent studies have shown that WW domain-binding protein 2 (WBP2) is important for the oncogenic property of breast cancer. WWP2 N-terminal-like (WBP2NL) is a testis-specific signaling protein that induces meiotic resumption and oocyte activation events. Our previous study revealed that WBP2NL gene expression is elevated in actively dividing cells and it might be associated with cellular proliferation and tumorigenic process. However, the clinical relevance and importance of WBP2NL gene in cancer has not been understood yet. Therefore, we were interested in analyzing the expression of WBP2NL gene in human breast cancer tissues and breast cancer cell lines, for the first time. We used reverse transcription-polymerase chain reaction (RT-PCR) and semi-nested RT-PCR to evaluate the expression of WBP2NL in malignant breast cancer and adjacent noncancerous tissue (ANCT) samples, as well as MCF-7 and MDA-MB-231 cell lines. The WBP2NL gene was expressed in 45 out of 50 (90%) breast cancer tissues and overexpressed in the MDA-MB-231 cell line. We suggest that WBP2NL may play roles in breast cancer activation maybe through binding to a group I WW domain protein. The elevated expression of WBP2NL gene in breast cancer and MDA-MB-231 cell line leads us to suggest that WBP2NL might be considered as a novel prognostic factor for early diagnosis of breast cancer.