NEK2 promotes the development of ovarian endometriosis and impairs decidualization by phosphorylating FOXO1.

Ovarian endometriosis is a common gynecological disease, and one of its most significant symptoms is infertility. In patients with endometriosis, defects in endometrial decidualization lead to impaired endometrial receptivity and embryo implantation, thus affecting early pregnancy and women's desire to have children. However, the mechanisms underlying the development of endometriosis and its associated defective decidualization are unclear. We find that NEK2 expression is increased in the ectopic and eutopic endometrium of patients with endometriosis. Meanwhile, NEK2 interacts with FOXO1 and phosphorylates FOXO1 at Ser184, inhibiting the stability of the FOXO1 protein. Importantly, NEK2-mediated phosphorylation of FOXO1 at Ser184 promotes cell proliferation, migration, invasion and impairs decidualization. Furthermore, INH1, an inhibitor of NEK2, inhibits the growth of ectopic lesions in mouse models of endometriosis and promotes endometrial decidualization in mouse models of artificially induced decidualization. Taken together, these findings indicate that NEK2 regulates the development of endometriosis and associated disorders of decidualization through the phosphorylation of FOXO1, providing a new therapeutic target for its treatment.

CHIP induces ubiquitination and degradation of HMGB1 to regulate glycolysis in ovarian endometriosis.

Ovarian endometriosis is a common gynecological condition that can cause infertility in women of childbearing age. However, the pathogenesis is still unknown. We demonstrate that the carboxyl terminus of Hsc70-interacting protein (CHIP) is a negative regulator in the development of endometriosis and reduces HMGB1 expression in endometriotic cells. Meanwhile, CHIP interacts with HMGB1 and promotes its ubiquitinated degradation, thereby inhibiting aerobic glycolysis and the progression of endometriosis. Furthermore, the CHIP agonist YL-109 effectively suppresses the growth of ectopic endometrium in endometriosis mouse model, which could be a potential therapeutic approach for endometriosis. In conclusion, our data suggest that CHIP may inhibit the development of endometriosis by suppressing the HMGB1-related glycolysis.

The effect of flexible low-dose GnRH antagonist on pregnancy outcome in the fresh embryo transfer cycle of IVF-ET: a randomized controlled trial.

OBJECTIVE: To explore the practicality and effectiveness of a flexible low-dose protocol in the fresh embryo transfer cycle: reducing the total amount of antagonist by increasing the interval between administrations of Cetrotide. METHODS: A total of 211 patients with normal ovarian reserve who accepted GnRH-ant protocol for IVF-ET were selected, and they were randomized to the flexible low-dose antagonist group (test group, n = 101) or the conventional dose antagonist group (control group, n = 110). The initial dose of Cetrotide in the test group was 0.25 mg every other day, and then the dose was adjusted to 0.25 mg every day based on the subsequent luteinizing hormone (LH) levels. The dosage of Cetrotide in the control group was 0.25 mg per day. The primary outcome was the clinical pregnancy rate. Secondary outcomes included the incidence of premature LH rise, total dosage of Cetrotide, number of oocytes retrieved, number of fertilized oocytes, number of high-quality embryos, biochemical pregnancy rate and ongoing pregnancy rate. RESULTS: There was no significant difference in the general condition of the two groups. There was no significant difference in the clinical pregnancy rate (51.49% vs. 48.18%, p = 0.632) or the incidence of premature LH rise (18.81% vs. 15.45%, p = 0.584) between the two groups. However, the amount of Cetrotide used in the test group was significantly lower than that in the conventional dose antagonist group (1.13 ± 0.41 vs. 1.61 ± 0.59 mg, p < 0.001). CONCLUSION: The flexible low-dose antagonist protocol and the conventional dose antagonist protocol were equally effective in people with a normal ovarian reserve in the fresh embryo transfer cycle of IVF-ET.

PKM2 Thr454 phosphorylation increases its nuclear translocation and promotes xenograft tumor growth in A549 human lung cancer cells.

Pyruvate kinase M2 (PKM2) is a key enzyme of glycolysis which is highly expressed in many tumor cells, and plays an important role in the Warburg effect. In previous study, we found PIM2 phosphorylates PKM2 at Thr454 residue (Yu, etl 2013). However, the functions of PKM2 Thr454 modification in cancer cells still remain unclear. Here we find PKM2 translocates into the nucleus after Thr454 phosphorylation. Replacement of wild type PKM2 with a mutant (T454A) enhances mitochondrial respiration, decreases pentose phosphate pathway, and enhances chemosensitivity in A549 cells. In addition, the mutant (T454A) PKM2 reduces xenograft tumor growth in nude mice. These findings demonstrate that PKM2 T454 phosphorylation is a potential therapeutic target in lung cancer.

The association between the number of pregnancies and depressive symptoms: A population-based study.

BACKGROUND: Depression is a psychosomatic disorder that affects reproductive health. The number of pregnancies is an important indicator of reproductive health. Multiple pregnancies and births may aggravate the risk of depression in females. However, the evidence of the connection between the number of pregnancies and depression is unclear. We aimed to investigate the relationship between the number of pregnancies and depressive symptoms. METHODS: We used the National Health and Nutrition Examination Survey (NHANES) data with a total of 17,216 women from 2005 to 2020. The number of pregnancies obtained from the self-report questionnaire. Depressive symptoms were measured by the nine-item patient health questionnaire (PHQ-9). Multivariate logistic regression models were used to examine the risk factors of depression. The restricted cubic spline (RCS) was applied to explore the nonlinear relationship. In addition, subgroup analysis was used to support the accuracy of our findings. RESULTS: We found that the number of pregnancies is positively associated with the prevalence of depression. According to the multivariable logistic regression analysis, pregnant women was 1.52-fold higher than the normal group to experience depression in the fully-adjusted model. No interaction between number of pregnancies and covariates in subgroups. LIMITATIONS: This study was cross-sectional, which limits its ability to draw conclusions about the causal relationship between the number of pregnancies and depression. CONCLUSION: In the United States, the number of pregnancies was positively associated with the prevalence of depression. It is critical to register the number of pregnancies for monitoring depressive symptoms.

SARS-CoV-2 infection negatively impacts on the quality of embryos by delaying early embryonic development.

BACKGROUND: The COVID-19 pandemic is an unprecedented health crisis that has affected in vitro fertilization practices globally. Previous studies have shown that SARS-CoV-2 impacts the quality of embryos by inducing an immunological response in infertile patients. In this study, the early embryonic development of SARS-CoV-2-infected infertile patients was investigated. METHODS: Sixty-five SARS-CoV-2 infected infertile patients and 258 controls were involved in this study. The major outcome parameters for the cycle were analyzed, including the number of oocytes, maturation oocytes, available embryos per cycle, and embryo morpho kinetic characteristics. RESULTS: From SARS-CoV-2 infection until oocyte retrieval, it took an average of 6.63 days. The results revealed that the number of oocytes and high-quality embryos on day 3 dramatically reduced in SARS-CoV-2-infected infertile patients. SARS-CoV-2 was detected in the follicular fluid of three infertile patients. SARS-CoV-2 infection had negatively impacted the number of oocytes in multivariate linear regression models. The early embryonic development in the SARS-CoV-2 infection group had a noticeable delay from the six-cell stage to blastocyst stage. CONCLUSIONS: SARS-CoV-2 infection reduced the number of oocytes and high-quality embryos on day 3. It delays the early embryonic development from the six-cell stage to blastocyst stage and has a negative impact on the quality of embryos.

Single-cell transcriptomics reveals male germ cells and Sertoli cells developmental patterns in dairy goats.

Spermatogenesis holds considerable promise for human-assisted reproduction and livestock breeding based on stem cells. It occurs in seminiferous tubules within the testis, which mainly comprise male germ cells and Sertoli cells. While the developmental progression of male germ cells and Sertoli cells has been widely reported in mice, much less is known in other large animal species, including dairy goats. In this study, we present the data of single cell RNA sequencing (scRNA-seq) for 25,373 cells from 45 (pre-puberty), 90 (puberty), and 180-day-old (post-puberty) dairy goat testes. We aimed to identify genes that are associated with key developmental events in male germ cells and Sertoli cells. We examined the development of spermatogenic cells and seminiferous tubules from 15, 30, 45, 60, 75, 90, 180, and 240-day-old buck goat testes. scRNA-seq clustering analysis of testicular cells from pre-puberty, puberty, and post-puberty goat testes revealed several cell types, including cell populations with characteristics of spermatogonia, early spermatocytes, spermatocytes, spermatids, Sertoli cells, Leydig cells, macrophages, and endothelial cells. We mapped the timeline for male germ cells development from spermatogonia to spermatids and identified gene signatures that define spermatogenic cell populations, such as AMH, SOHLH1, INHA, and ACTA2. Importantly, using immunofluorescence staining for different marker proteins (UCHL1, C-KIT, VASA, SOX9, AMH, and PCNA), we explored the proliferative activity and development of male germ cells and Sertoli cells. Moreover, we identified the expression patterns of potential key genes associated with the niche-related key pathways in male germ cells of dairy goats, including testosterone, retinoic acid, PDGF, FGF, and WNT pathways. In summary, our study systematically investigated the elaborate male germ cells and Sertoli cells developmental patterns in dairy goats that have so far remained largely unknown. This information represents a valuable resource for the establishment of goat male reproductive stem cells lines, induction of germ cell differentiation in vitro, and the exploration of sequential cell fate transition for spermatogenesis and testicular development at single-cell resolution.

Deubiquitination of MYC by OTUB1 contributes to HK2 mediated glycolysis and breast tumorigenesis.

MYC as a transcriptional factor plays a crucial role in breast cancer progression. However, the mechanisms underlying MYC deubiquitination in breast cancer are not well defined. Here, we report that OTUB1 is responsible for MYC deubiquitination. OTUB1 could directly deubiquitinate MYC at K323 site, which blocks MYC protein degradation. Moreover, OTUB1 mediated MYC protein stability is also confirmed in OTUB1-knockout mice. Stabilized MYC by OTUB1 promotes its transcriptional activity and induces HK2 expression, which leads to enhance aerobic glycolysis. Therefore, OTUB1 promotes breast tumorigenesis in vivo and in vitro via blocking MYC protein degradation. Taken together, our data identify OTUB1 as a new deubiquitination enzyme for MYC protein degradation, which provides a potential target for breast cancer treatment.

Ubiquitination of NF-κB p65 by FBXW2 suppresses breast cancer stemness, tumorigenesis, and paclitaxel resistance.

The F-box and WD-repeat-containing protein 2 (FBXW2) plays a crucial role as an E3 ligase in regulating tumorigenesis. However, the functions of FBXW2 in breast cancer are still unknown. Here, we find that nuclear factor-kB (NF-κB) p65 is a new substrate of FBXW2. FBXW2 directly binds to p65, leading to its ubiquitination and degradation. Interestingly, p300 acetylation of p65 blocks FBXW2 induced p65 ubiquitination. FBXW2-p65 axis is a crucial regulator of SOX2-induced stemness in breast cancer. Moreover, FBXW2 inhibits breast tumor growth by regulating p65 degradation in vitro and in vivo. FBXW2 overexpression abrogates the effects of p65 on paclitaxel resistance in vitro and in vivo. Furthermore, FBXW2 induced p65 degradation is also confirmed in FBXW2-knockout mice. Our results identify FBXW2 as an important E3 ligase for p65 degradation, which provide insights into the tumor suppressor functions of FBXW2 in breast cancer.

Phosphorylation of PFKFB4 by PIM2 promotes anaerobic glycolysis and cell proliferation in endometriosis.

Endometriosis (EM) is one of the vanquished wonted causes of chronic pelvic sting in women and is closely associated with infertility. The long-term, complex, systemic, and post-treatment recurrence of EM wreaks havoc on women's quality of life. Extensive metabolic reprogramming (aerobic glycolysis, glucose overweening intake, and high lactate production) and cancer-like changes have been found in EM, which bears striking similarities to tumorigenesis. The key glycolysis regulator PFKFB4 is overexpressed in EM. However, the mechanism of PFKFB4 in EM remains unknown. We found that PFKFB4 was upregulated and was closely related to the progression of EM. We identified focus PIM2 as a new pioneering adjoin protein of PFKFB4. Vigorous biochemical methods were used to confirm that PIM2 phosphorylated site Thr140 of PFKFB4. PIM2 also could enhance PFKFB4 protein expression through the ubiquitin-proteasome pathway. Moreover, PIM2 expression was really corresponding prevalent with PFKFB4 in endometriosis in vivo. Importantly, phosphorylation of PFKFB4 on Thr140 by PIM2 promoted EM glycolysis and cell growth. Our study demonstrates that PIM2 mediates PFKFB4 Thr140 phosphorylation thus regulating glycolysis and EM progression. We illustrated a new mechanism that PIM2 simulated a central upstream partnership in the regulation of PFKFB4, and reveal a novel means of PIM2-PFKFB4 setting EM growth. Our research provided new theoretical support for further clarifying the reprogramming of EM glucose metabolism, and provided new clues for exploring non-contraceptive treatments for EM.

Positive regulation of PFKFB3 by PIM2 promotes glycolysis and paclitaxel resistance in breast cancer.

BACKGROUND: Breast cancer (BC) is one of the most common female malignancies in the world. Chemotherapeutic resistance is the major cause of BC therapy failure, leading to tumor recurrence and metastasis. Studies have illustrated the close relationship between glycolysis and BC progression and drug resistance. The key glycolysis regulator, PFKFB3 makes a difference during BC progression and drug resistance. However, the mechanism remains to be unknown. METHODS: Mass spectrometry analyses were used to found that PIM2 was a potential new binding protein of PFKFB3. Co-immunoprecipitated and western blot were used to verify the interaction between PIM2 and PFKFB3 in BC and the molecular mechanism by which PIM2 phosphorylates PFKFB3 in regulating the protein function. PFKFB3 mutant forms were used to demonstrate the need for PFKFB3 in BC drug resistance. RESULTS: We identified that PIM2 is a new binding protein of PFKFB3. We used biochemical methods to determine that PIM2 can directly bind and change the phosphorylation of PFKFB3 at Ser478 to enhance PFKFB3 protein stability through the ubiquitin-proteasome pathway. Importantly, phosphorylation of PFKFB3 at Ser478 promoted glycolysis, BC cell growth, and paclitaxel resistance together with PIM2 in vitro and in vivo. CONCLUSION: Our study demonstrates that PIM2 mediates PFKFB3 phosphorylation thus regulates glycolysis and paclitaxel resistance to promote tumor progression in BC and provides preclinical evidence for targeting PFKFB3 as a new strategy in BC treatment to battle paclitaxel resistance.

Uterine natural killer cells and recurrent spontaneous abortion.

Recurrent spontaneous abortion (RSA), termed as two or more consecutive pregnancy loss is a great problem for some women of childbearing age. A large number of evidence confirm that there may be an immune background of RSA. As a member of the innate immune system, uterine natural killer (uNK) cells account for about 70% of total lymphocytes during pregnancy and play a critical role in the establishment and maintenance of pregnancy. This review mainly introduces the phenotype, origin, receptor, and function of uNK cells to illuminate its relationship with RSA.

Fructose-1, 6-bisphosphatase 1 interacts with NF-κB p65 to regulate breast tumorigenesis via PIM2 induced phosphorylation.

Rationale: Fructose-1, 6-bisphosphatase 1 (FBP1), a rate-limiting enzyme in gluconeogenesis, was recently shown to be a tumor suppressor and could mediate the activities of multiple transcriptional factors via its non-canonical functions. However, the underlying mechanism of posttranscriptional modification on the non-canonical functions of FBP1 remains elusive. Methods: We employed immunoaffinity purification to identify binding partner(s) and used co-immunoprecipitation to verify their interactions. Kinase reaction was used to confirm PIM2 could phosphorylate FBP1. Overexpression or knockdown proteins were used to assess the role in modulating p65 protein stability. Mechanistic analysis was involved in protein degradation and polyubiquitination assays. Nude mice and PIM2-knockout mice was used to study protein functions in vitro and in vivo. Results: Here, we identified Proviral Insertion in Murine Lymphomas 2 (PIM2) as a new binding partner of FBP1, which could phosphorylate FBP1 on Ser144. Surprisingly, phosphorylated FBP1 Ser144 abrogated its interaction with NF-κB p65, promoting its protein stability through the CHIP-mediated proteasome pathway. Furthermore, phosphorylation of FBP1 on Ser144 increased p65 regulated PD-L1 expression. As a result, phosphorylation of FBP1 on Ser144 promoted breast tumor growth in vitro and in vivo. Moreover, the levels of PIM2 and pSer144-FBP1 proteins were positively correlated with each other in human breast cancer and PIM2 knockout mice. Conclusions: Our findings revealed that phosphorylation noncanonical FBP1 by PIM2 was a novel regulator of NF-κB pathway, and highlights PIM2 inhibitors as breast cancer therapeutics.

Correction: PIM2-mediated phosphorylation of hexokinase 2 is critical for tumor growth and paclitaxel resistance in breast cancer.

A correction to this paper has been published and can be accessed via a link at the top of the paper.

A Noncanonical Role of Fructose-1, 6-Bisphosphatase 1 Is Essential for Inhibition of Notch1 in Breast Cancer.

Breast cancer is a leading cause of death in women worldwide, but the underlying mechanisms of breast tumorigenesis remain unclear. Fructose-1, 6-bisphosphatase 1 (FBP1), a rate-limiting enzyme in gluconeogenesis, was recently shown to be a tumor suppressor in breast cancer. However, the mechanisms of FBP1 as a tumor suppressor in breast cancer remain to be explored. Here we showed that FBP1 bound to Notch1 in breast cancer cells. Moreover, FBP1 enhanced ubiquitination of Notch1, further leading to proteasomal degradation via FBXW7 pathway. In addition, we found that FBP1 significantly repressed the transactivation of Notch1 in breast cancer cells. Functionally, Notch1 was involved in FBP1-mediated tumorigenesis of breast cancer cells in vivo and in vitro. Totally, these findings indicate that FBP1 inhibits breast tumorigenesis by regulating Notch1 pathway, highlighting FBP1 as a potential therapeutic target for breast cancer. IMPLICATIONS: We demonstrate FBP1 as a novel regulator for Notch1 in breast cancer.

Negative regulation of AMPKα1 by PIM2 promotes aerobic glycolysis and tumorigenesis in endometrial cancer.

Endometrial cancer (EC) is one of the most common gynecologic malignancies. However, the molecular mechanisms underlying the development and progression of EC remain unclear. Here, we demonstrated that the protein proviral insertion in murine lymphomas 2 (PIM2) was necessary for maintaining EC tumorigenesis in vivo and in vitro, and could inhibit AMPKα1 kinase activity in EC cells. Specifically, we found that PIM2 bound to AMPKα1, and directly phosphorylated it on Thr467. Phosphorylation of AMPKα1 by PIM2 led to decreasing AMPKα1 kinase activity, which in turn promoted aerobic glycolysis and tumor growth. In addition, PIM2 expression positively correlated with AMPKα1 Thr467 phosphorylation in EC tissues. Further, treatment with a combination of the PIM2 inhibitor SMI-4a and the AMPKα1 activator AICAR could effectively inhibit tumor growth. Thus, our findings provide insight into the role of PIM2 and AMPKα1 in EC and suggest that combination targeting of these proteins may represent a new strategy for EC treatment.

Phosphorylation of HSF1 by PIM2 Induces PD-L1 Expression and Promotes Tumor Growth in Breast Cancer.

Heat shock transcription factor 1 (HSF1) is the master regulator of the proteotoxic stress response, which plays a key role in breast cancer tumorigenesis. However, the mechanisms underlying regulation of HSF1 protein stability are still unclear. Here, we show that HSF1 protein stability is regulated by PIM2-mediated phosphorylation of HSF1 at Thr120, which disrupts the binding of HSF1 to the E3 ubiquitin ligase FBXW7. In addition, HSF1 Thr120 phosphorylation promoted proteostasis and carboplatin-induced autophagy. Interestingly, HSF1 Thr120 phosphorylation induced HSF1 binding to the PD-L1 promoter and enhanced PD-L1 expression. Furthermore, HSF1 Thr120 phosphorylation promoted breast cancer tumorigenesis in vitro and in vivo. PIM2, pThr120-HSF1, and PD-L1 expression positively correlated with each other in breast cancer tissues. Collectively, these findings identify PIM2-mediated HSF1 phosphorylation at Thr120 as an essential mechanism that regulates breast tumor growth and potential therapeutic target for breast cancer. SIGNIFICANCE: These findings identify heat shock transcription factor 1 as a new substrate for PIM2 kinase and establish its role in breast tumor progression.

PIM2-mediated phosphorylation of hexokinase 2 is critical for tumor growth and paclitaxel resistance in breast cancer.

Hexokinase-II (HK2) is a key enzyme involved in glycolysis, which is required for breast cancer progression. However, the underlying post-translational mechanisms of HK2 activity are poorly understood. Here, we showed that Proviral Insertion in Murine Lymphomas 2 (PIM2) directly bound to HK2 and phosphorylated HK2 on Thr473. Biochemical analyses demonstrated that phosphorylated HK2 Thr473 promoted its protein stability through the chaperone-mediated autophagy (CMA) pathway, and the levels of PIM2 and pThr473-HK2 proteins were positively correlated with each other in human breast cancer. Furthermore, phosphorylation of HK2 on Thr473 increased HK2 enzyme activity and glycolysis, and enhanced glucose starvation-induced autophagy. As a result, phosphorylated HK2 Thr473 promoted breast cancer cell growth in vitro and in vivo. Interestingly, PIM2 kinase inhibitor SMI-4a could abrogate the effects of phosphorylated HK2 Thr473 on paclitaxel resistance in vitro and in vivo. Taken together, our findings indicated that PIM2 was a novel regulator of HK2, and suggested a new strategy to treat breast cancer.