ASB3 promotes hepatocellular carcinoma progression by mediating DR5 ubiquitination in TRAIL resistance.

Ankyrin-repeat proteins with a suppressor of cytokine signaling box (ASB) proteins belong to the E3 ubiquitin ligase family. 18 ASB members have been identified whose biological functions are mostly unexplored. Here, we discovered that ASB3 was essential for hepatocellular carcinoma (HCC) development and high ASB3 expression predicted poor clinical outcomes. ASB3 silencing induced HCC cell growth arrest and apoptosis in vitro and in vivo. Liver-specific deletion of Asb3 gene suppressed diethylnitrosamine (DEN)-induced liver cancer development. Mechanistically, ASB3 interacted with death receptor 5 (DR5), which promoted ubiquitination and degradation of DR5. We further showed that ASB3 knockdown stabilized DR5 and increased the sensitivity of liver cancer cells to the treatment of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in a DR5-dependent manner in cellular and in animal models. In summary, we demonstrated that ASB3 promoted ubiquitination and degradation of DR5 in HCC, suggesting the potential of targeting ASB3 to HCC treatment and overcome TRAIL resistance.

MST1/2: Important regulators of Hippo pathway in immune system associated diseases.

The Hippo signaling pathway is first found in Drosophila and is highly conserved in evolution. Previous studies on this pathway in mammals have revealed its key role in cell proliferation and differentiation, organ size control, and carcinogenesis. Apart from these, recent findings indicate that mammalian Ste20-like kinases 1 and 2 (MST1/2) have significant effects on immune regulation. In this review, we summarize the updated understanding of how MST1/2 affect the regulation of the immune system and the specific mechanism. The effect of MST1/2 on immune cells and its role in the tumor immune microenvironment can alter the body's response to tumor cells. The relationship between MST1/2 and the immune system suggests new directions in the manipulation of immune responses for clinical immunotherapy, especially for tumor treatment.

CBX4 deletion promotes tumorigenesis under KrasG12D background by inducing genomic instability.

Chromobox protein homolog 4 (CBX4) is a component of the Polycomb group (PcG) multiprotein Polycomb repressive complexes 1 (PRC1), which is participated in several processes including growth, senescence, immunity, and tissue repair. CBX4 has been shown to have diverse, even opposite functions in different types of tissue and malignancy in previous studies. In this study, we found that CBX4 deletion promoted lung adenocarcinoma (LUAD) proliferation and progression in KrasG12D mutated background. In vitro, over 50% Cbx4L/L, KrasG12D mouse embryonic fibroblasts (MEFs) underwent apoptosis in the initial period after Adeno-Cre virus treatment, while a small portion of survival cells got increased proliferation and transformation abilities, which we called selected Cbx4-/-, KrasG12D cells. Karyotype analysis and RNA-seq data revealed chromosome instability and genome changes in selected Cbx4-/-, KrasG12D cells compared with KrasG12D cells. Further study showed that P15, P16 and other apoptosis-related genes were upregulated in the primary Cbx4-/-, KrasG12D cells due to chromosome instability, which led to the large population of cell apoptosis. In addition, multiple pathways including Hippo pathway and basal cell cancer-related signatures were altered in selected Cbx4-/-, KrasG12D cells, ultimately leading to cancer. We also found that low expression of CBX4 in LUAD was associated with poorer prognosis under Kras mutation background from the human clinical data. To sum up, CBX4 deletion causes genomic instability to induce tumorigenesis under KrasG12D background. Our study demonstrates that CBX4 plays an emerging role in tumorigenesis, which is of great importance in guiding the clinical treatment of lung adenocarcinoma.

Generation of Vgll4-DreER transgenic mouse for visualizing and manipulating VGLL4-expressing cells in vivo.

Vestigial like family member 4 (VGLL4), a member of the Hippo pathway, is a transcriptional cofactor involved in many biological processes, such as tumor progression, postnatal heart growth, and muscle regeneration. However, the VGLL4 expression pattern in vivo remains unclear. To detect and trace Vgll4-expressing cells and their progeny, we generated and characterized a new tamoxifen-inducible Dre knock-in mouse line, Vgll4-DreER. This mouse line expressed DreER (Dre recombinase fused to the estrogen receptor) under the control of the endogenous Vgll4 promoter. After crossing the Vgll4-DreER mouse line with the Dre-responsive reporter H11-rRFP, Dre-mediated recombination in the tissue was monitored on the basis of red fluorescent protein (RFP) signals, which indicated the distribution of VGLL4-positive cells in vivo. Our data revealed that VGLL4 is widely expressed in various cell types at embryonic and neonatal stages. After comparison with our previously reported Vgll4-GFP mouse, we found that the RFP signal profile was wider than the green fluorescent protein (GFP) pattern, indicating that Vgll4-DreER is more sensitive for labeling VGLL4-expressing cells. We next used a dual-recombination system to simultaneously label VGLL4- and keratin 5 (KRT5)-positive cell populations, and no crosstalk was observed in the Krt5-CreER;Vgll4-DreER;R26-rGlR mice. Taken together, the Vgll4-DreER mouse line is a valuable new tool for examining the precise VGLL4 expression profile and conditional manipulating of VGLL4-expressing cells and their progeny.

Hippo Pathway in Schwann Cells and Regeneration of Peripheral Nervous System.

Hippo pathway is an evolutionarily conserved signaling pathway comprising a series of MST/LATS kinase complexes. Its key transcriptional coactivators YAP and TAZ regulate transcription factors such as TEAD family to direct gene expression. The regulation of Hippo pathway, especially the nuclear level change of YAP and TAZ, significantly influences the cell fate switching from proliferation to differentiation, regeneration, and postinjury repair. This review outlines the main findings of Hippo pathway in peripheral nerve development, regeneration, and tumorigenesis, especially the studies in Schwann cells. We also summarize other roles of Hippo pathway in damage repair of the peripheral nerve system and discuss the potential future research which probably contributes to novel therapeutic strategies.

Sleep Disturbance Affects Immune Factors in Clinical Liver Cancer Patients.

BACKGROUND: Sleep-wake disturbance is prevalent in patients with liver cancer, but there is no direct evidence of its association and related biological mechanisms. Our study was to assess quality of sleep and to describe prevalence of sleep disturbances in patients with different etiologies of liver cancer, especially to explore whether sleep quality influences immune factors. METHODS: A total of 210 patients with liver cancer from August 2015 to December 2015 were randomly divided into two groups including HBV cirrhosis and non-HBV cirrhosis. The Pittsburgh Sleep Quality Index (PSQI) was used to evaluate their sleep quality, and then 202 patients enrolled in this study were divided into two groups according to their PSQI scores: PSQI ≤ 5 and PSQI > 5. The association between sleep disturbances and immune factors was analyzed by logistic regression models. RESULTS: A total of 56.9% of liver cancer patients experienced poor sleep quality (PSQI > 5). The prevalence of sleep disturbances was significantly higher in patients with liver cancer of non-hepatitis B virus (HBV) cirrhosis than with that evolving from HBV cirrhosis (66.7% vs. 50%, p = 0.018). In non-HBV cirrhosis liver cancer patients, the PSQI > 5 group had a higher percentage of CD3+ T cells (71.06 ± 11.07 vs. 63.96 ± 14.18, p = 0.014) and lower natural killer (NK) cells (14.67 ± 9.65 vs. 20.5 ± 10.77, p = 0.014) compared with patients with PSQI ≤ 5. Logistic regression further confirmed that liver cancer patients without HBV cirrhosis are more prone to experience poor sleep with increased CD3+ T cells (OR = 1.07, 95% CI = 1.01-1.13, p = 0.030) and decreased NK cells (OR = 0.92, 95% CI = 0.85-0.98, p = 0.014). Our results indicate that increased CD3+ T cells and decreased NK cells are both associated with sleep disturbances in patients with liver cancer of non-HBV cirrhosis. CONCLUSIONS: Most liver cancer patients suffer from sleep disturbances, especially evolving from non-HBV cirrhosis. A rise in CD3+ T cells and a reduction in NK cells are associated with sleep disturbances in patients with liver cancer of non-HBV cirrhosis.

Selective YAP activation in Procr cells is essential for ovarian stem/progenitor expansion and epithelium repair.

Ovarian surface epithelium (OSE) undergoes recurring ovulatory rupture and OSE stem cells rapidly generate new cells for the repair. How the stem cell activation is triggered by the rupture and promptly turns on proliferation is unclear. Our previous study has identified that Protein C Receptor (Procr) marks OSE progenitors. In this study, we observed decreased adherent junction and selective activation of YAP signaling in Procr progenitors at OSE rupture site. OSE repair is impeded upon deletion of Yap1 in these progenitors. Interestingly, Procr+ progenitors show lower expression of Vgll4, an antagonist of YAP signaling. Overexpression of Vgll4 in Procr+ cells hampers OSE repair and progenitor proliferation, indicating that selective low Vgll4 expression in Procr+ progenitors is critical for OSE repair. In addition, YAP activation promotes transcription of the OSE stemness gene Procr. The combination of increased cell division and Procr expression leads to expansion of Procr+ progenitors surrounding the rupture site. These results illustrate a YAP-dependent mechanism by which the stem/progenitor cells recognize the murine ovulatory rupture, and rapidly multiply their numbers, highlighting a YAP-induced stem cell expansion strategy.

Chromobox 4 facilitates tumorigenesis of lung adenocarcinoma through the Wnt/ß-catenin pathway.

Chromobox 4 (CBX4) is a core component of polycomb-repressive complex 1 with important roles in cancer biology and tissue homeostasis. Aberrant expression of CBX4 has been implicated in several human malignancies. However, its role and underlying mechanisms in the tumorigenesis of lung adenocarcinoma (LUAD) have not been defined in vivo. Here, we found that expression of CBX4 was frequently up-regulated in human LUAD samples and correlated with poor patient survival. Importantly, genetic ablation of CBX4 greatly dampened lung tumor formation and improved survival in the KrasG12D/P53L/L (KP) autochthonous mouse model of LUAD. In addition, CBX4 depletion significantly inhibited proliferation and anchorage-independent growth of KP mouse embryonic fibroblasts. Moreover, ectopic CBX4 expression clearly promoted proliferation and anchorage-independent growth in both human and mouse LUAD cells, whereas silencing of CBX4 exerted opposite effects. Mechanistically, CBX4 promoted growth of LUAD cells through activation of the Wnt/ß-catenin pathway. Furthermore, expression levels of CBX4 were positively correlated with ß-catenin in human LUAD samples. In conclusion, our data suggest that CBX4 plays an oncogenic role via the Wnt/ß-catenin pathway and could serve as a potential therapeutic target in LUAD.

VGLL4 promotes osteoblast differentiation by antagonizing TEADs-inhibited Runx2 transcription.

VGLL4 has been identified as a YAP inhibitor. However, the exact function of VGLL4 in bone development and bone homeostasis remains unclear. In this study, we demonstrated that VGLL4 breaks TEADs-mediated transcriptional inhibition of RUNX2 to promote osteoblast differentiation and bone development. We found that knockout of VGLL4 in mesenchymal stem cells and preosteoblasts showed osteoporosis and a cleidocranial dysplasia-like phenotype due to osteoblast differentiation disorders. Mechanistically, we showed that the TEAD transcriptional factors severely inhibited osteoblast differentiation in a YAP binding-independent manner. TEADs interacted with RUNX2 to repress RUNX2 transcriptional activity. Furthermore, VGLL4 relieved the transcriptional inhibition of TEADs by directly competing with RUNX2 to bind TEADs through its two TDU domains. Collectively, our studies demonstrate that VGLL4 plays an important role in regulating osteoblast differentiation and bone development, and that TEADs regulate the transcriptional activity of RUNX2, which may shed light on treatment of cleidocranial dysplasia and osteoporosis.

A Regulation Loop between YAP and NR4A1 Balances Cell Proliferation and Apoptosis.

The Hippo signaling pathway maintains organ size and tissue homeostasis via orchestration of cell proliferation and apoptosis. How this pathway triggers cell apoptosis remains largely unexplored. Here, we identify NR4A1 as a target of the Hippo pathway that mediates the pro-apoptotic and anti-tumor effects of the Hippo pathway whereby YAP regulates the transcription, phosphorylation, and mitochondrial localization of NR4A1. NR4A1, in turn, functions as a feedback inhibitor of YAP to promote its degradation, thereby inhibiting the function of YAP during liver regeneration and tumorigenesis. Our studies elucidate a regulatory loop between NR4A1 and YAP to coordinate Hippo signaling activity during liver regeneration and tumorigenesis and highlight NR4A1 as a marker of Hippo signaling, as well as a therapeutic target for hepatocellular carcinoma.

The Tumor Suppressor Interferon Regulatory Factor 2 Binding Protein 2 Regulates Hippo Pathway in Liver Cancer by a Feedback Loop in Mice.

BACKGROUND AND AIMS: The conserved Hippo pathway regulates organ size, tissue homeostasis, and tumorigenesis. Interferon regulatory factor 2 binding protein 2 (IRF2BP2) was originally identified as a transcriptional corepressor. However, the association between IRF2BP2 and the Hippo pathway remains largely unknown. In addition, the biological function and regulation mechanism of IRF2BP2 in liver cancer are poorly understood. APPROACH AND RESULTS: In this study, we uncovered the clinical significance of IRF2BP2 in suppressing hepatocellular carcinogenesis. We showed that IRF2BP2, a direct target repressed by the Yes-associated protein (YAP)/TEA domain transcription factor 4 (TEAD4) transcriptional complex, inhibited YAP activity through a feedback loop. IRF2BP2 stabilized vestigial-like family member 4 (VGLL4) and further enhanced VGLL4's inhibitory function on YAP. Moreover, liver-specific IRF2BP2 overexpression suppressed tumor formation induced by Hippo pathway inactivation. CONCLUSIONS: These results revealed the important role of IRF2BP2 in repressing liver cancer progression and highlighted a feedback loop underlying the Hippo pathway in organ-size control and tumorigenesis.

Glucocorticoid Receptor Signaling Activates TEAD4 to Promote Breast Cancer Progression.

The Hippo pathway plays a critical role in cell growth and tumorigenesis. The activity of TEA domain transcription factor 4 (TEAD4) determines the output of Hippo signaling; however, the regulation and function of TEAD4 has not been explored extensively. Here, we identified glucocorticoids (GC) as novel activators of TEAD4. GC treatment facilitated glucocorticoid receptor (GR)-dependent nuclear accumulation and transcriptional activation of TEAD4. TEAD4 positively correlated with GR expression in human breast cancer, and high expression of TEAD4 predicted poor survival of patients with breast cancer. Mechanistically, GC activation promoted GR interaction with TEAD4, forming a complex that was recruited to the TEAD4 promoter to boost its own expression. Functionally, the activation of TEAD4 by GC promoted breast cancer stem cells maintenance, cell survival, metastasis, and chemoresistance both in vitro and in vivo. Pharmacologic inhibition of TEAD4 inhibited GC-induced breast cancer chemoresistance. In conclusion, our study reveals a novel regulation and functional role of TEAD4 in breast cancer and proposes a potential new strategy for breast cancer therapy. SIGNIFICANCE: This study provides new insight into the role of glucocorticoid signaling in breast cancer, with potential for clinical translation.

Cell Division Cycle 42 plays a Cell type-Specific role in Lung Tumorigenesis.

Cell division cycle 42 (CDC42) plays important roles in polarity establishment and maintenance as well as cell cycle progression and cell division. Although disruption of cell polarity is a prerequisite in epithelial tumor initiation, the roles of CDC42 in tumorigenesis are still poorly understood. Here we find that Cdc42 deficiency inhibits the Kras G12D -induced lung alveoli tumor formation, while conversely promotes bronchiole tumor formation in mice. Bronchial Cdc42 loss destroys contact inhibition potentially through cell polarity disruption, and results in increased tumor formation. In contrast, deletion of Cdc42 in alveoli cells prevents Kras G12D -induced cell proliferation, which leads to reduced tumor formation. Further analyses of clinical specimens uncover a significant positive correlation between CDC42 and type II alveolar epithelial cells marker SP-A, indicating the potential importance of CDC42 in this specific subset of lung cancer. Collectively, we identify the lineage-specific function of CDC42 in lung tumorigenesis potentially through the regulation of cell polarity integrity.

A novel BMX variant promotes tumor cell growth and migration in lung adenocarcinoma.

The non-receptor tyrosine kinase BMX has been reported in several solid tumors. However, the alternative splicing of BMX and its clinical relevance in lung cancer remain to be elucidated. Exon1.0 array was used to identify a novel alternative splicing of BMX, BMXΔN, which was confirmed by rapid amplification of cDNA ends and reverse transcription-polymerase chain reaction. BMXΔN, resulting from exon skipping with excluding exon 1 to exon 8 of BMX gene, was found in 12% human lung adenocarcinoma specimens. BMXΔN is not found in paired pathologically normal lungs and positively correlated with EGFR mutation in lung adenocarcinomas. Moreover, BMXΔN increases cell proliferation, neoplastic transformation, and migratory property of human non-small cell lung cancer cells. The function of BMXΔN in lung cancer might be presumably due to enhanced ERK signaling.

Identification of TRA2B-DNAH5 fusion as a novel oncogenic driver in human lung squamous cell carcinoma.

Lung squamous cell carcinoma (SCC) is one of the major subtypes of lung cancer. Our current knowledge of oncogenic drivers in this specific subtype of lung cancer is largely limited compared with lung adenocarcinoma (ADC). Through exon array analyses, molecular analyses and functional studies, we here identify the TRA2B-DNAH5 fusion as a novel oncogenic driver in lung SCC. We found that this gene fusion occurs exclusively in lung SCC (3.1%, 5/163), but not in lung ADC (0/119). Through mechanistic studies, we further revealed that this TRA2B-DNAH5 fusion promotes lung SCC malignant progression through regulating a SIRT6-ERK1/2-MMP1 signaling axis. We show that inhibition of ERK1/2 activation using selumetinib efficiently inhibits the growth of lung SCC with TRA2B-DNAH5 fusion expression. These findings improve our current knowledge of oncogenic drivers in lung SCC and provide a potential therapeutic strategy for lung SCC patients with TRA2B-DNAH5 fusion.

Tumor-secreted miR-214 induces regulatory T cells: a major link between immune evasion and tumor growth.

An increased population of CD4(+)CD25(high)Foxp3(+) regulatory T cells (Tregs) in the tumor-associated microenvironment plays an important role in cancer immune evasion. However, the underlying mechanism remains unclear. Here we observed an increased secretion of miR-214 in various types of human cancers and mouse tumor models. Tumor-secreted miR-214 was sufficiently delivered into recipient T cells by microvesicles (MVs). In targeted mouse peripheral CD4(+) T cells, tumor-derived miR-214 efficiently downregulated phosphatase and tensin homolog (PTEN) and promoted Treg expansion. The miR-214-induced Tregs secreted higher levels of IL-10 and promoted tumor growth in nude mice. Furthermore, in vivo studies indicated that Treg expansion mediated by cancer cell-secreted miR-214 resulted in enhanced immune suppression and tumor implantation/growth in mice. The MV delivery of anti-miR-214 antisense oligonucleotides (ASOs) into mice implanted with tumors blocked Treg expansion and tumor growth. Our study reveals a novel mechanism through which cancer cell actively manipulates immune response via promoting Treg expansion.

VGLL4 functions as a new tumor suppressor in lung cancer by negatively regulating the YAP-TEAD transcriptional complex.

Lung cancer is one of the most devastating diseases worldwide with high incidence and mortality. Hippo (Hpo) pathway is a conserved regulator of organ size in both Drosophila and mammals. Emerging evidence has suggested the significance of Hpo pathway in cancer development. In this study, we identify VGLL4 as a novel tumor suppressor in lung carcinogenesis through negatively regulating the formation of YAP-TEAD complex, the core component of Hpo pathway. Our data show that VGLL4 is frequently observed to be lowly expressed in both mouse and human lung cancer specimens. Ectopic expression of VGLL4 significantly suppresses the growth of lung cancer cells in vitro. More importantly, VGLL4 significantly inhibits lung cancer progression in de novo mouse model. We further find that VGLL4 inhibits the activity of the YAP-TEAD transcriptional complex. Our data show that VGLL4 directly competes with YAP in binding to TEADs and executes its growth-inhibitory function through two TDU domains. Collectively, our study demonstrates that VGLL4 is a novel tumor suppressor for lung cancer through negatively regulating the YAP-TEAD complex formation and thus the Hpo pathway.

Two co-existing germline mutations P53 V157D and PMS2 R20Q promote tumorigenesis in a familial cancer syndrome.

Germline mutations are responsible for familial cancer syndromes which account for approximately 5-10% of all types of cancers. These mutations mainly occur at tumor suppressor genes or genome stability genes, such as DNA repair genes. Here we have identified a cancer predisposition family, in which eight members were inflicted with a wide spectrum of cancer including one diagnosed with lung cancer at 22years old. Sequencing analysis of tumor samples as well as histologically normal specimens identified two germline mutations co-existing in the familial cancer syndrome, the mutation of tumor suppressor gene P53 V157D and mismatch repair gene PMS2 R20Q. We further demonstrate that P53 V157D and/or PMS2 R20Q mutant promotes lung cancer cell proliferation. These two mutants are capable of promoting colony formation in soft agar as well as tumor formation in transgenic drosophila system. Collectively, these data have uncovered the important role of co-existing germline P53 and PMS2 mutations in the familial cancer syndrome development.