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YAP promotes ROS accumulation via the DNp63-GPX2 signaling axis

YAP activation inhibits SCC cell growth by disturbing ROS homeostasis.

Negative correlation of YAP and P63 expression in human lung SCC.

Lung squamous cell carcinoma (SCC), accounting for approximately 30% of non–small cell lung cancer, is often refractory to therapy. Screening a small-molecule library, we identified digitoxin as a high potency compound for suppressing human lung SCC growth in vitro and in vivo. Mechanistic investigations revealed that digitoxin attenuated YAP phosphorylation and promoted YAP nuclear sequestration. YAP activation led to excessive accumulation of reactive oxygen species (ROS) by downregulating the antioxidant enzyme GPX2 in a manner related to p63 blockade. In patient-derived xenograft models, digitoxin treatment efficiently inhibited lung SCC progression in correlation with reduced expression of YAP. Collectively, our results highlight a novel tumor-suppressor function of YAP via downregulation of GPX2 and ROS accumulation, with potential implications to improve precision medicine of human lung SCC. Cancer Res; 77(21); 5769–81. ©2017 AACR.

Digitoxin treatment in mice doesn't result in significant weight loss and pathological or immunochemical changes.

Lung squamous cell carcinoma (SCC), accounting for approximately 30% of non–small cell lung cancer, is often refractory to therapy. Screening a small-molecule library, we identified digitoxin as a high potency compound for suppressing human lung SCC growth in vitro and in vivo. Mechanistic investigations revealed that digitoxin attenuated YAP phosphorylation and promoted YAP nuclear sequestration. YAP activation led to excessive accumulation of reactive oxygen species (ROS) by downregulating the antioxidant enzyme GPX2 in a manner related to p63 blockade. In patient-derived xenograft models, digitoxin treatment efficiently inhibited lung SCC progression in correlation with reduced expression of YAP. Collectively, our results highlight a novel tumor-suppressor function of YAP via downregulation of GPX2 and ROS accumulation, with potential implications to improve precision medicine of human lung SCC. Cancer Res; 77(21); 5769–81. ©2017 AACR.

Several immune checkpoint blockers have been approved for treatment of certain cancers, which benefits a lot of patients with malignancies. However, the overall response rate to the current checkpoint blockers did not exceed 30% in many cases, stressing the importance of developing new immunotherapies. Siglec-15 is reported to be a critical immune suppressor functioning in parellel with PD1/PD-L1 pathway by Lieping Chen's lab in 2019. In order to verify the effect Siglec-15 in immune response against tumor growth, we generated Siglec-15 KO mice with CRISPR/Cas9 mediated genome editing and tested tumor establishment on this mice with syngeneic cell lines with or without human SIGLEC-15 overexpression. Our results showed that Siglec-15 KO inhibits tumor growth and enhances immune response in the tumor infiltrating leukocytes, supporting the role of Siglec-15 as a promising target for development of new immune checkpoint blocker. Citation Format: Peixue Li, Guofang Yan, Fuyang Wang, Xuzhen Tang, Qing Lin. Siglec-15 knockout inhibits tumor growth in mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1634.

The volatiles of Lindera glauca (Sieb. et Zucc.) fruits with different maturities, grown in Jigongshan mountain, Henan China, were isolated by hydro-distillation method and analyzed by GC-qMS. Eigh...

Epidermal Growth Factor Receptor (EGFR) overexpression is observed in a large subset of patients with non-small-cell lung cancer (NSCLC) and correlated with poor prognosis. Current approved treatment for EGFR mutation-positive NSCLC has been revolutionized with three generations of EGFR tyrosine kinase inhibitors (TKIs): the first- and second-generation TKIs, such as gefitinib, erlotinib and afatinib, selectively targeting EGFR activating mutations existing in exon 19 and 21; and the third-generation EGFR-wild-type sparing, irreversible activating mutations/T790M inhibitor, osimertinib. Patients with somatic activating mutations in the EGFR gene have dramatic response initially, but would eventually develop resistance to these TKIs. One leading evidence of osimertinib resistance is mediated by occurrence of the point mutation C797S. There are no effective therapeutic strategies to overcome this triple-mutation (activating mutations/T790M/C797S) mediated resistance. Therefore, it is urged to develop new drug to target this particular mutant. There is still lack of triple-mutation in vitro model for the new generation drug discovery. In this study, a triple-mutation NSCLC model was generated in the NCI-H1975 lung cancer cell line, which harbors other naturally occurring EGFR genomic aberrations - double-mutation (T790M/L858R) - inherent in NSCLC. We utilized the CRISPR/Cas9 genome editing tool to target endogenous loci in human NSCLC cell line NCI-H1975 and created the intend point mutation, resulting in the triple mutation cell line (T790M/C797S/L858R). Further phenotypic analysis demonstrated that the triple-mutation cell line was resistant to the third generation inhibitors in contrast to the parental double-mutation cell line. This newly developed triple-mutation lung cancer cell line provided a very useful tool for oncology drug discovery for NSCLC. Citation Format: Huiying Ma, Peixue Li, Peiran Zhang, Fuyun Sun, Qing Lin. Generation of a triple-mutation (T790M/C797S/L858R) NSCLC cell line for relevant drug discovery and development [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1864.

It has been challenging for efficient gene editing in cells which are hard to transfect, such as stem cells and primary T cells. Typical sgRNA and Cas9 intracellular delivery techniques are limited by their reliance on cell type and exogenous materials as well as their toxic effects on cells (for example, electroporation) or packaging capacity limitation by viral vectors. Here we established an effective approach for gene editing in different cell types and achieved successful genome editing with CRISPR RNP electroporation. Also, high efficient double knockout mutations could be obtained by electroporation of multiple sgRNAs at one shot. Furthermore, this method also could reduce the exposure time to Cas9 nuclease which may cause potential off-target effects. With the advantages of broad applicability across different cell types, particularly hard-to-transfect cells, and flexibility of application, these methods could potentially enable the development of the stem cell or T cell based therapies. Citation Format: Peixue Li, Zhao Li, Qing Lin. CRISPR/Cas9 RNP mediated efficient gene editing in hard-to-transfect cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6303.

We investigate deep learning based omni intrusion detection system (IDS) for supervisory control and data acquisition (SCADA) networks that are capable of detecting both temporally uncorrelated and correlated attacks. Regarding the IDSs developed in this paper, a feedforward neural network (FNN) can detect temporally uncorrelated attacks at an {F$_{1}$} of {99.967${\pm}$0.005\%} but correlated attacks as low as {58${\pm}$2\%}. In contrast, long-short term memory (LSTM) detects correlated attacks at {99.56${\pm}$0.01\%} while uncorrelated attacks at {99.3${\pm}$0.1\%}. Combining LSTM and FNN through an ensemble approach further improves the IDS performance with {F$_{1}$} of {99.68${\pm}$0.04\%} regardless the temporal correlations among the data packets.

We present recurrent neural networks (RNN) for supervisory control and data acquisition (SCADA) Intrusion Detection System (IDS). Using long short term memory (LSTM) with many-to-many (MTM) and a novel many-to-one (MTO) architectures, both IDSs display excellent performance in detecting temporal uncorrelated attacks while MTO showing superior performance on temporal correlated attacks.

The Hippo pathway plays a stage-specific role in regeneration and fibrogenesis after ischaemia/reperfusion-induced acute kidney injury. The proper modulation of this pathway might be the key point of transition from acute kidney injury to chronic kidney disease., Renal tubule cells can recover after they undergo AKI (acute kidney injury). An incomplete repair of renal tubules can result in progressive fibrotic CKD (chronic kidney disease). Studies have revealed the relationship between tubular epithelial cells and kidney fibrogenesis. However, the underlying mechanism remains unclear. Hippo pathway components were evaluated in complete/incomplete repair of I/R (ischaemia/reperfusion) AKI rat models, HK-2 cells and AKI human renal biopsy samples. We found that the expression levels of the Hippo pathway components changed dynamically during kidney regeneration and fibrogenesis in rat models of I/R-induced AKI and human renal biopsy samples. The transcription cofactor YAP (Yes-associated protein) might be a key effector of renal regeneration and fibrogenesis. Our results showed further that YAP might elicit both beneficial and detrimental effects on I/R AKI. After I/R injury occurred, YAP could promote the repair of the injured epithelia. The constant YAP increase and activation might be related to interstitial fibrosis and abnormal renal tubule differentiation. These results indicate that the proper modulation of the Hippo pathway, specifically the transcription cofactor YAP, during repair might be a potent therapeutic target in AKI–CKD transition after I/R injury.

The evolutionarily conserved Hippo signaling pathway plays an important role in organ size control by regulating cell proliferation and apoptosis. Here, we identify Lingerer (Lig) as a growth suppressor using RNAi modifying screen in Drosophila melanogaster. Loss of lig increases organ size and upregulates bantam (ban) and the expression of the Hippo pathway target genes, while overexpression of lig results in diminished ban expression and organ size reduction. We demonstrate that Lig C-terminal exhibits dominant-negative function on growth and ban expression, and thus plays an important role in organ size control and ban regulation. In addition, we provide evidence that both Yki and Mad are essential for Lig-induced ban expression. We also show that Lig regulates the expression of the Hippo pathway target genes partially via Yorkie. Moreover, we find that Lig physically interacts with and requires Salvador to restrict cell growth. Taken together, we demonstrate that Lig functions as a critical growth suppressor to control organ size via ban and Hippo signaling.

To investigate the effects of multiple environmental conditions on greenhouse gas (CO2 , N2 O, CH4 ) fluxes, we transferred three soil monoliths from Masson pine forest (PF) or coniferous and broadleaved mixed forest (MF) at Jigongshan to corresponding forest type at Dinghushan. Greenhouse gas fluxes at the in situ (Jigongshan), transported and ambient (Dinghushan) soil monoliths were measured using static chambers. When the transported soil monoliths experienced the external environmental factors (temperature, precipitation and nitrogen deposition) at Dinghushan, its annual soil CO2 emissions were 54% in PF and 60% in MF higher than those from the respective in situ treatment. Annual soil N2 O emissions were 45% in PF and 44% in MF higher than those from the respective in situ treatment. There were no significant differences in annual soil CO2 or N2 O emissions between the transported and ambient treatments. However, annual CH4 uptake by the transported soil monoliths in PF or MF was not significantly different from that at the respective in situ treatment, and was significantly lower than that at the respective ambient treatment. Therefore, external environmental factors were the major drivers of soil CO2 and N2 O emissions, while soil was the dominant controller of soil CH4 uptake. We further tested the results by developing simple empirical models using the observed fluxes of CO2 and N2 O from the in situ treatment and found that the empirical models can explain about 90% for CO2 and 40% for N2 O of the observed variations at the transported treatment. Results from this study suggest that the different responses of soil CO2 , N2 O, CH4 fluxes to changes in multiple environmental conditions need to be considered in global change study.

Masson pine (Pinus massoniana Lamb.), a native species widely distributed in temperate forests in central China, and Loblolly pine (Pinus taeda L.), an exotic tree species introduced to China from southeastern United States, are dominant evergreen conifers that play a pivotal role in maintaining forest structure and functions for the region. We examined the effects of freezing on these species with chlorophyll fluorescence and electrolyte leakage using both field- and laboratory-based experiments in September 2009 and January 2010, respectively. We found that freezing could cause a greater impact on the Loblolly pine than the Masson pine. Although the two species showed similar values of Fv/Fm and electrolyte leakage before freezing, the Masson pine needles showed lower Fv/Fm and higher electrolyte leakage ratios than those of the Loblolly pine when treated in low temperatures (-15 to 0C). We also found that cold-acclimation was crucial for both species to adapt to low temperatures with the Fv/Fm ratio decreased approximately by 80% in the first freezing hour for the non-acclimated needles of both species while the cold-acclimated needles showed little changes in the Fv/Fm ratio. This finding is also supported by our measurements of electrolyte leakage. These results suggest that the Loblolly pine could be more susceptible to freezing damages than the Masson pine in central China. 2012 Copyright Taylor and Francis Group, LLC.

This work reveals the formation mechanisms of saturation dislocation patterns in three typical multiple-slip oriented [0 0 1], [0 1 1] and ½ 111 � copper single crystals. Compared with the single-slip oriented copper single crystals, the three multiple-slip oriented ones show very different dislocation patterns. It was found that the dislocation patterns in cyclically saturated copper single crystals are the Labyrinth structure for [0 0 1], wall structure for [0 1 1] and cell structure for ½ 111 � , respectively. Based on a two-phase structure consisting of persistent slip bands and veins for single-slip orientation, the formation mechanisms of the dislocation patterns in multiple-slip oriented crystals are proposed as follows: the formation of the complex dislocation patterns depends on the activating slip system. The easy operation of the critical secondary slip system will contribute to the formation of the Labyrinth structure. The activation of the coplanar secondary slip system will be beneficial to formation of the cell structure. If no secondary slip system is activated, the wall structure is more prone to appear. Finally, the intrinsic relationship between various dislocation patterns and face centered cubic crystal structure was established.

Fragile X syndrome (FraX), the most common form of inherited mental retardation, is caused by the absence of the evolutionally conserved fragile X mental retardation protein (FMRP). While neuronal functions of FMRP have been intensively studied for the last two decades, its role in non-neuronal cells remains poorly understood. Piwi, a key component of the Piwi-interacting RNA (piRNA) pathway, plays an essential role in germline development. In the present study, we report that similar to piwi, dfmr1, the Drosophila homolog of human FMR1, is required for transposon suppression in the germlines. Genetic analyses showed that dfmr1 and piwi act synergistically in heterochromatic silencing, and in inhibiting the differentiation of primordial germline cells and transposon expression. Northern analyses showed that roo piRNA expression levels are reduced in dfmr1 mutant ovaries, suggesting a role of dfmr1 in piRNA biogenesis. Biochemical analysis demonstrated a physical interaction between dFMRP and Piwi via their N-termini. Taken together, we propose that dFMRP cooperates with Piwi in maintaining genome integrity by regulating heterochromatic silencing in somatic cells and suppressing transposon activity via the piRNA pathway in germlines.

Increasing atmospheric nitrogen (N) deposition could profoundly impact community structure and ecosystem functions in forests. However, conventional experiments with understory addition of N (UAN) largely neglect canopy-associated biota and processes and therefore may not realistically simulate atmospheric N deposition to generate reliable impacts on forest ecosystems. Here we, for the first time, designed a novel experiment with canopy addition of N (CAN) vs. UAN and reviewed the merits and pitfalls of the two approaches. The following hypotheses will be tested: i) UAN overestimates the N addition effects on understory and soil processes but underestimates those on canopy-associated biota and processes, ii) with low-level N addition, CAN favors canopy tree species and canopy-dwelling biota and promotes the detritus food web and iii) with high-level N addition, CAN suppresses canopy tree species and other biota and favors rhizosphere food web. As a long-term comprehensive program, this experiment will provide opportunities for multidisciplinary collaborations, including biogeochemistry, microbiology, zoology and plant science to examine forest ecosystem responses to atmospheric N deposition.

Pez functions as an upstream negative regulator of Yorkie (Yki) to regulate intestinal stem cell (ISC) proliferation and is essential for the activity of the Hippo pathway specifically in the Drosophila midgut epithelium. Here we report that Suppressor of Deltex (Su(dx)) acts as a negative regulator of Pez. We show that Su(dx) targets Pez for degradation both in vitro and in vivo. Overexpression of Su(dx) induces proliferation in the fly midgut epithelium, which can be rescued by overexpressed Pez. We also demonstrate that the interaction between Su(dx) and Pez, bridged by WW domains and PY/PPxY motifs, is required for Su(dx)-mediated Pez degradation. Furthermore, we find that Kibra, a binding partner of Pez, stabilizes Pez via WW-PY/PPxY interaction. Moreover, PTPN14, a Pez mammalian homolog, is degraded by overexpressed Su(dx) or Su(dx) homologue WWP1 in mammalian cells. These results reveal a previously unrecognized mechanism of Pez degradation in maintaining the homeostasis of Drosophila midgut.

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.

Quantitative real time reverse transcription polymerase chain reaction has been applied in a vast range of studies of gene expression analysis. However, real-time PCR data must be normalized with one or more reference genes. In this study, eleven putative consistently expressed genes (ACT, TUA, TUB, CYP, DNAj, ELFA, F-box27, RPL12, GAPDH, UBC and UBQ) in nine Siberian Apricot Germplasms (including much variability) were evaluated for their potential as references for the normalization of gene expression by NormFinder and geNorm programs. From our studies, ACT, UBC, CYP, UBQ and RPL12 as suitable for normalization were identified by geNorm, while UBC and CYP as the best pair by NormFinder. Moreover, UBC was selected as the most stably expressed gene by both algorithms in different Siberian Apricot seed samples. We also detected that a set of three genes (ACT, CYP and UBC) by geNorm as control for normalization could lead to accurate results. Furthermore, the expression levels of oleosin gene were analyzed to validate the suitability of the selected reference genes. These obtained experimental results could make an important contribution to normalize real-time PCR data for gene expression analysis in Siberian Apricot Germplasm.

The Hippo (Hpo) pathway controls tissue growth and organ size by regulating the activity of transcriptional co-activator Yorkie (Yki), which associates with transcription factor Scalloped (Sd) in the nucleus to promote downstream target gene expression. Here we identify a novel protein Sd-Binding-Protein (SdBP)/Tgi, which directly competes with Yki for binding to Sd through its TDU domains and inhibits the Sd-Yki transcriptional activity. We also find that SdBP retains Yki in the nucleus through the association with Yki WW domains via its PPXY motifs. Collectively, we identify SdBP as a novel component of the Hpo pathway, negatively regulating the transcriptional activity of Sd-Yki to restrict tissue growth.

SummaryThe Hippo pathway has been implicated in suppressing tissue overgrowth and tumor formation by restricting the oncogenic activity of YAP. However, transcriptional regulators that inhibit YAP activity have not been well studied. Here, we uncover clinical importance for VGLL4 in gastric cancer suppression and find that VGLL4 directly competes with YAP for binding TEADs. Importantly, VGLL4’s tandem Tondu domains are not only essential but also sufficient for its inhibitory activity toward YAP. A peptide mimicking this function of VGLL4 potently suppressed tumor growth in vitro and in vivo. These findings suggest that disruption of YAP-TEADs interaction by a VGLL4-mimicking peptide may be a promising therapeutic strategy against YAP-driven human cancers.