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Epidermal growth factor receptor (EGFR) mutation is prevalently expressed in lung adenocarcinoma cases and acts as one of the major driving oncogenes. EGFR tyrosine kinase inhibitors (TKIs) have been used in patients with EGFR-mutant as an effective targeted therapy in lung adenocarcinoma, but drug resistance and tumor recurrence inevitably occurs. Recently, Yes-associate protein (YAP) has been reported to promote multiple cancer cell properties, such as promoting cell proliferation, epithelial-mesenchymal transition and drug resistance. This study investigated the roles of YAP in TKI-resistant lung adenocarcinoma. In TKI-sensitive cells, enhanced YAP expression leads to TKI resistant. Also, upregulated YAP expression and activation were detected in long-term TKI-induced resistant cells. With reduced YAP expression using shRNA or YAP inhibitors, TKI-resistant cells become TKI-sensitive. reduced xenograft tumor size in nude mice and Moreover, combined EGFR TKI and a YAP inhibitor, statin, prolonged survival among lung cancer patients analyzed by Taiwan National Health Insurance Research database. These observations revealed the importance of YAP in promoting TKI-resistance and combined YAP inhibition can be a potential therapy delaying the occurrence of TKI-resistance in lung adenocarcinoma.

Lung cancer is the leading cause of cancer death, and therefore the discovery of novel therapeutic targets is crucial. P21-activated kinase (PAK1) is an important oncogene involved in the signaling of actin cytoskeleton organization. Although PAK1 inhibition has been shown to suppress cancer progression, specific PAK1 inhibitors are not available due to the complex structure and insufficient understanding of this kinase. The Hippo signaling effector TAZ is known to be elevated in multiple human cancers and to promote cancer metastasis. This study aimed to explore the role of TAZ in regulating the tumor suppressor ankyrin repeat domain 52 (ANKRD52) and PAK1 activity. A negative correlation between TAZ and ANKRD52 was observed, with knockdown of TAZ leading to enhanced ANKRD52 promoter activity and increased mRNA levels. Moreover, reduced ANKRD52 levels were associated with late-stage lung cancer. Knockdowns of ANKRD52 resulted in elevated cell mobility, while forced ANKRD52 expression attenuated cell mobility. ANKRD52 is a subunit of the protein phosphatase 6 (PP6) holoenzyme. Mass spectrometry analysis revealed the interaction between PAK1 and the ANKRD52-PP6 complex. Knockdown of ANKRD52 or PP6c resulted in upregulated PAK1 phosphorylation. Our study demonstrates that the novel tumor suppressor protein ANKRD52 is transcriptionally inhibited by TAZ, regulating cell mobility through interactions with PP6c and dephosphorylation of PAK1.

// Ting-Fang Lee 1 , Yu-Chi Tseng 2 , Wei-Chin Chang 1,3 , Yi-Chen Chen 2 , Yu-Rung Kao 4 , Teh-Ying Chou 1,5 , Chao-Chi Ho 6 and Cheng-Wen Wu 1,2,4 1 Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan 2 Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan 3 Department of Pathology, MacKay Memorial Hospital, Taipei, Taiwan 4 Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 5 Department of Pathology, Taipei Veterans General Hospital, Taipei, Taiwan 6 Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University Medical College, Taipei, Taiwan Correspondence to: Cheng-Wen Wu, email: // Keywords : YAP1, EGFR mutation, lung adenocarcinoma, TKI-resistance Received : December 21, 2016 Accepted : June 20, 2017 Published : July 27, 2017 Abstract Epidermal growth factor receptor (EGFR) mutations are found in lung adenocarcinomas leading to tumor cells proliferation and survival. EGFR tyrosine kinase inhibitors (TKIs) that block EGFR activity are effective therapeutics for EGFR-mutant lung adenocarcinoma patients, but TKI-resistance inevitably occurs. The YES-associated protein (YAP1) transcription coactivator has been implicated as an oncogene and is amplified in human cancers and provides tumor cells strong proliferation and survival cues. This study investigated the roles of YAP1 in lung adenocarcinoma by exploring its regulation and functions mediated by EGFR signaling. In this study, we detected a correlation between YAP1 level and EGFR mutation status in lung adenocarcinoma tissues. Using lung adenocarcinoma cell lines, enhanced YAP1 expression and activity mediated by EGFR signaling was detected through enhanced protein stability. A SRC family protein, YES, was involved in EGFR-regulated YAP1 expression and this pathway was crucial for proliferation in EGFR-dependent cells. Small molecules that reduced YAP1 levels by mechanisms bypassing EGFR signaling were effective in reducing viability in EGFR-dependent cells including those with EGFR T790M, the major cause of TKI-resistance. These observations unveiled the significance of YAP1 in EGFR mutant lung adenocarcinomas and identified YAP1 as a promising therapeutic target for EGFR-dependent lung adenocarcinoma patients, including those with EGFR T790M-caused TKI resistance.

PAK1 is an important oncogene involved in the progression of a variety of cancer types including lung cancer. Although PAK1 inhibition is shown to suppress tumor progression, specific PAK1 inhibitors are not available due to the complex structure and insufficient understanding of this protein. In an attempt to find TAZ (WW domain-containing transcription regulator 1) regulated tumor suppressor, a member of ankyrin repeat proteins, ankyrin repeat domain 52 (ANKRD52), is observed to suppress PAK1 phosphorylation. Ankyrin repeat proteins are known to function as a platform for protein-protein interactions, and ANKRD52 was shown to interact with protein phosphatase 6 (PP6). Immunohistochemistry staining in lung cancer tissue revealed prolonged disease-free survival in high ANKRD52 expressing cases. Using lung cancer cell lines, ANKRD52 knockdowns showed significantly increased cell mobility while forced expressed ANKRD52 decreased cell mobility. Using mass spectrometry analysis, p21-activated kinases (PAK1) was identified to interact with ANKRD52. Upregulated PAK1 phosphorylation was detected in the knockdowns of ANKRD52 or PP6. Our model demonstrated a novel anti-metastatic protein, ANKRD52, that regulates cell mobility through interactions with PP6 and dephosphorylation of PAK1. Citation Format: Ting-Fang Lee, Yin-Pu Liu, Yen-Fan Lin, Chong-Fang Hsu, Cheng-Wen Wu. ANKRD52 inhibited tumor metastasis through dephosphorylation of PAK1 in lung adenocarcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1782.

Liver sinusoids are lined by a fenestrated endothelium that lacks a basement membrane. Formation of perisinusoidal basement membranes beneath the endothelium is an integral feature of capillarization of sinusoids that is a significant pathology found in advanced fibrosis. Liver fibrosis is prevalent in elderly cadavers; however, basement membrane formation in these liver samples has yet to be studied. Collagen type IV and laminin are major basement membrane proteins and their codistribution around sinusoids provides an immunohistochemical marker of basement membrane formation. Here, we examined the intralobular sites of perisinusoidal basement membrane formation in elderly cadaveric livers having various stages of fibrosis. Collagen IV and laminin codistributed in basement membranes of portal and septal ductular and vascular structures, providing a positive control. In the parenchyma, collagen IV immunostaining of sinusoids was panlobular in all stages of fibrosis, and the stain was continuous along the sinusoids. In contrast, laminin was not detected in livers, showing minimal fibrotic change. It was rarely seen in perisinusoidal/pericellular fibrosis, but frequently in septa formation, bridging fibrosis, and cirrhosis. The laminin stain was patchy, occurring principally in sinusoids of periportal and periseptal areas, less commonly in mid-lobular and rarely in centrilobular areas. Consecutive sections revealed that laminin codistributed with collagen IV in these sinusoidal locations, thus marking the sites of perisinusoidal basement membrane formation in aged fibrotic livers. This development is presumably related to aging of the liver and exacerbated by liver injury caused by advanced liver fibrosis, possibly resulting in sinusoidal capillarization.

Eur J Clin Invest 2012; 42 (6): 607–616 Abstract Background We previously demonstrated that kaerophyllin, a lignan, isolated from a widely used traditional Chinese herb, Bupleurum scorzonerifolium, leading to the inhibition of hepatic stellate cells (HSCs) activation in vitro. This current study evaluated the in vivo role of kaerophyllin in protecting the liver against injury and fibrogenesis caused by thioacetamide (TAA) in rats and further explored the underlying mechanisms. Materials and methods Liver fibrosis in Sprague-Dawley rats was induced by intraperitoneal injection of TAA (200 mg/kg) twice per week for 6 weeks. Animals were divided into five groups: vehicle control, TAA control, TAA + low dose kaerophyllin, TAA + high dose kaerophyllin and TAA + curcumin groups. Kaerophyllin (10 or 30 mg/kg) or curcumin (150 mg/mL) was given by gavage twice per day consecutively for 4 weeks starting 2 weeks after TAA injection. Rat HSCs were used to investigate the anti-inflammatory role of kaerophyllin against tumour necrosis factor α (TNF-α) in vitro. Peroxisome proliferator-activated receptor-γ (PPAR-γ) expression was knocked down in rat HSCs using PPAR-γ small interfering RNAs. Results Kaerophyllin significantly protected liver from injury by reducing serum aspartate transaminase and alanine transaminase levels and by improving the histological architecture and fibrosis score. In addition, kaerophyllin suppressed inflammation by reducing the mRNA of TNF-α, interleukin-1β (IL-1β) and monocyte chemoattractant protein-1 (MCP-1) genes. In HSCs, kaerophyllin elevated PPAR-γ activity and reduced TNF-α-stimulated mRNA levels of intracellular adhesion molecule-1 (ICAM-1), MCP-1 and IL-1β genes, which were reversed by small interfering RNA knockdown of PPAR-γ gene. Conclusions Our results demonstrated that kaerophyllin protected the rat liver from TAA-caused injury and fibrogenesis by suppressing hepatic inflammation and inhibiting HSC activation, possibly through upregulation of PPAR-γ expression.

Background: Hepatic stellate cells (HSCs), the key cell type for hepatic fibrosis, become activated and profibrogenic in the presence of hepatocyte apoptotic bodies (ABs). Bupleurum scorzonerifolium (BS), a widely used traditional Chinese herb for liver diseases, was fractionated, and the inhibitory effects of BS extracts on AB-induced HSC migration were screened. The activity-guided fractionation led to a lignan, kaerophyllin. In this study, the anti-fibrotic effects of kaerophyllin were studied in the presence of ABs. Methods: LX-2 cells phagocytosing ultraviolet (UV)-induced HepG2 ABs were investigated by confocal microscopy and flow cytometry. AB-induced HSC activation was evaluated by immunoblotting and real-time PCR analyses. HSC migrationwas measured by wound-healing assays. Results: HepG2 ABs induced LX-2 activation, with the production of collagen I and a-smooth muscle actin, upregulated profibrogenic gene transcriptions and increased NF-kB activity, cell migration and phagocytosis. Kaerophyllin from BS antagonized ABinduced HSC migration and activation. Conclusions: Kaerophyllin inhibited AB-induced LX-2 activation and migration with downregulation of Akt/ERK phosphorylations and NF-kB activity. Our study suggests a novel platform for screening anti-fibrotic compounds with ABs.

Cancer metastasis is an ultimate challenge in our effort to fight cancer as a life-threatening disease. A member of ankyrin repeat proteins, ankyrin repeat domain 52 (ANKRD52), is studied here to be involved in the regulation of lung adenocarcinoma metastasis. Ankyrin repeat proteins are known to function as a platform for protein-protein interactions, and ANKRD52 was shown to interact with protein phosphatase 6 (PP6). Immunohistochemistry staining in lung adenocarcinomas tissues revealed low ANKRD52 expressing in stage-IV patients compared to those in stages I to III. Overexpressed ANKRD52 prevented lung cancer formation in the xenograft model. Using lung cancer cell lines, ANKRD52 knockdowns showed significantly increased cell mobility while forced expression decreased cell mobility. Using mass spectrometry analysis, p21-activated kinases (PAK1) was identified to interact with ANKRD52. The regulation of PAK1 phosphorylation by the ANKRD52-PP6 complex is essential to cell mobility. Our model demonstrated a novel anti-metastatic protein, ANKRD52, that regulates cell mobility through interacting with PP6 leading to PAK1 dephosphorylation. Citation Format: Ting-Fang Lee, Yen-Fan Lin, Ying-Pu Liu, Cheng-Wen Wu. ANKRD52 inhibited tumor metastasis in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5508.

Activation of the Hippo pathway effector Yap underlies many liver cancers, however no germline or somatic mutations have been identified. Autophagy maintains essential metabolic functions of the liver, and autophagy-deficient murine models develop benign adenomas and hepatomegaly, which have been attributed to activation of the p62/Sqstm1-Nrf2 axis. Here, we show that Yap is an autophagy substrate and mediator of tissue remodeling and hepatocarcinogenesis independent of the p62/Sqstm1-Nrf2 axis. Hepatocyte-specific deletion of Atg7 promotes liver size, fibrosis, progenitor cell expansion, and hepatocarcinogenesis, which is rescued by concurrent deletion of Yap. Our results shed new light on mechanisms of Yap degradation and the sequence of events that follow disruption of autophagy, which is impaired in chronic liver disease., Increased levels of the Yap oncoprotein stimulate liver growth and promote hepatocarcinogenesis. Here the authors show that hepatocyte-specific loss of Atg7 in mice leads to decreased autophagic degradation of Yap and liver overgrowth, and further establish this association in human liver cancer tissues.

Non-alcoholic steatohepatitis (NASH) is associated with hepatic fibrogenesis. Despite well-known cholesterol-lowering action of statins, their mechanisms against NASH-mediated fibrogenesis remain unclear. This study aimed at investigating the in vitro and in vivo anti-fibrotic properties of fluvastatin (Flu). Palmitate (PA)-induced changes in intracellular hydrogen peroxide levels in primary rat hepatocytes (PRHs) and human hepatoma cell line (HepG2) were quantified by dichlorofluorescein diacetate (DCF-DA) dye assay, whereas changes in expressions of NADPH oxidase gp91 phox subunit, α-smooth muscle actin (α-SMA), and NFκB p65 nuclear translocation were quantified with Western blotting. Quantitative real-time polymerase chain reaction (q-PCR) was used to investigate mRNA expressions of pro-inflammatory genes (ICAM-1, IL-6, TNF-α). Conditioned medium (CM) from PA-treated PRHs was applied to cultured rat hepatic stellate cell line, HSC-T6, with or without Flu-pretreatment for 2 h. Pro-fibrogenic gene expressions (COL1, TIMP-1, TGF-β1, α-SMA) and protein expression of α-SMA were analyzed. In vivo study using choline-deficient L-amino acid defined (CDAA) diet-induced rat NASH model was performed by randomly assigning Wistar rats (n = 28) to normal controls (n = 4), CDAA diet with vehicles, and CDAA diet with Flu (5 mg/kg or 10 mg/kg) (n = 8 each) through gavage for 4 or 8 weeks. Livers were harvested for histological, Western blot (α-SMA), and q-PCR analyses for expressions of pro-inflammatory (IL-6, iNOS, ICAM-1) and pro-fibrogenic (Col1, α-SMA, TIMP-1) genes. In vitro, Flu (1–20 μM) inhibited PA-induced free-radical production, gp91 phox expression, and NFκB p65 translocation in HepG2 and PRHs, while CM-induced α-SMA protein expression and pro-fibrogenic gene expressions in HSC-T6 were suppressed in Flu-pretreated cells compared to those without pretreatment. Moreover, α-SMA protein expression was significantly decreased in HSC-T6 cultured with CM from PA-Flu-treated PRHs compared to those cultured with CM from PA-treated PRHs. Flu also reduced steatosis and fibrosis scores, α-SMA protein expression, mRNA expression of pro-inflammatory and pro-fibrogenic genes in livers of CDAA rats. We demonstrated PA-induced HSC activation through paracrine effect of hepatocyte in vitro that was significantly suppressed by pre-treating HSC with Flu. In vivo, Flu alleviated steatosis-induced HSC activation and hepatic fibrogenesis through mitigating inflammation and oxidative stress, suggesting possible therapeutic role of Flu against NASH.

Background and Aims: Platelet-derived growth factor (PDGF) is a very potent mitogen for hepatic stellate cells (HSC) in hepatic fibrogenesis. Ligusticum chuanxiong Hort. (LC), a traditional Chinese herb used for cerebrovascular diseases, has been shown to exert anti-inflammatory and free radical scavenging effects. The aims of the present study were to investigate the effects of LC extract on the proliferation-related biomarkers in a rat HSC cell line (HSC-T6) stimulated with PDGF. Methods: DNA synthesis via bromodeoxyuridine (BrdU) incorporation, cell cycle related proteins and apoptosis markers were determined to evaluate the inhibitory effects of LC. Results: The results revealed that LC extract (25–100 µg/mL) concentration-dependently decreased the PDGF-induced cell proliferation as well as α-smooth muscle actin expression in HSC. The inhibitory activity of LC on HSC was associated with: (i) inhibition of BrdU incorporation; (ii) induction of apoptosis with the activation of caspase-3, up-regulation of cell cycle inhibitory proteins p21 and p27, and down-regulation of cell cycle stimulatory proteins cyclins D1 and D2; and (iii) increased phosphorylation of mitogen-activated protein kinases (JNK). LC at the studied concentrations showed no direct cytotoxicity on primary hepatocytes. Conclusion: The results suggest that LC significantly inhibited PDGF-activated HSC proliferation, possibly through apoptotic mechanisms and the potential of LC as an antifibrotic agent warrants further investigation.

As a leading cause of cancer-related deaths, metastasis is the ultimate challenge in our effort to fight cancer as a life-threatening disease. A member of ankyrin repeat proteins, ankyrin repeat domain 52 (ANKRD52), is studied here to be involved in the regulation of lung adenocarcinoma metastasis. Ankyrin repeat proteins are known to function as a platform for protein-protein interactions, and ANKRD52 was shown to interact with protein phosphatase 6 (PP6). This study aimed to investigate the anti-metastatic roles of ANKRD52 in lung adenocarcinoma. Immunohistochemistry staining in lung cancer tissue revealed prolonged disease-free survival in high ANKRD52 expressing cases. Using lung cancer cell lines, ANKRD52 knockdowns showed significantly increased cell mobility while forced expressed ANKRD52 decreased cell mobility. Using mass spectrometry analysis, p21-activated kinases (PAK1) was identified to interact with ANKRD52. Upregulated PAK1 phosphorylation was detected in ANKRD52 or PP6 knockdowns. Our model demonstrated a novel anti-metastatic protein, ANKRD52, that regulates cell mobility through interactions with PP6 and dephosphorylation of PAK1. Citation Format: Yen-Fan Lin, Ying-Pu Liu, Ting-Fang Lee, Cheng-Wen Wu. ANKRD52 inhibited tumor metastasis through dephosphorylation of PAK1 in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4843. doi:10.1158/1538-7445.AM2017-4843

Epidermal growth factor receptor (EGFR) mutation is prevalently expressed in lung adenocarcinoma cases and acts as one of the major driving oncogenes. EGFR tyrosine kinase inhibitors (TKIs) have been used in patients with EGFR-mutant as an effective targeted therapy in lung adenocarcinoma, but drug resistance and tumor recurrence inevitably occurs. Recently, Yes-associate protein (YAP) has been reported to promote multiple cancer cell properties, such as promoting cell proliferation, EMT and drug resistance. This study investigated the roles of YAP in TKI-resistant lung adenocarcinoma. In TKI-sensitive cells, enhanced YAP expression leads to TKI resistant. Also, upregulated YAP expression and activation were detected in long term TKI-induced resistant cells. With reduced YAP expression using shRNA or YAP inhibitors, TKI-resistant cells become TKI-sensitive. Moreover, combined EGFR TKI and an YAP inhibitor statin, prolonged survival among lung cancer patients analyzed by Taiwan National Health Insurance Research database. These observations revealed the importance of YAP in promoting TKI-resistance and combined YAP inhibition can be a potential therapy delaying the occurrence of TKI-resistance in lung adenocarcinoma. Citation Format: Ting-Fang Lee, Yu-Chi Tseng, Cheng-Wen Wu. Enhanced YAP expression leads to EGFR TKI resistance in lung adenocarcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1189. doi:10.1158/1538-7445.AM2017-1189

Epidermal growth factor receptor (EGFR) mutations are found in lung adenocarcinomas leading to tumor cells proliferation and survival. EGFR tyrosine kinase inhibitors (TKIs) that block EGFR activity are effective therapeutics for EGFR-mutant lung adenocarcinoma patients, but TKI-resistance inevitably occurs. The YES-associated protein (YAP) transcription coactivator has been implicated as an oncogene and is amplified in human cancers and provides tumor cells strong proliferation and survival cues. This study investigated the roles of YAP in lung adenocarcinoma by exploring its regulation and functions mediated by EGFR signaling. Using lung adenocarcinoma cell lines, we detected enhanced YAP expression and activity mediated by EGFR signaling due to enhanced protein stability. The Src family protein YES was involved in EGFR-regulated YAP expression. The YAP/YES pathway was crucial for proliferation in EGFR-dependent cells. Small molecules that reduced YAP levels by mechanisms bypassing EGFR were effective in killing EGFR-dependent cells including EGFR T790M, the major cause of TKI-resistance. These observations unveiled the significance of YAP in EGFR mutant lung adenocarcinomas and identified YAP as an promising therapeutic target for EGFR-dependent lung adenocarcinoma patients, including those with EGFR T790M-caused TKI resistance. Citation Format: Ting-Fang Lee, Yu-Chi Tseng, Ying-Pu Liu, Yu-Rung Kao, Cheng-Wen Wu. YAP is an important pathway downstream of EGFR that is a potential target for EGFR-TKI-resistance lung adenocarcinomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2146.

Suppression of hepatic stellate cell (HSC) growth and activation, and induction of apoptosis, have been proposed as therapeutic strategies for the treatment and prevention of liver fibrosis. Our previous study showed that the Chinese herb Ligusticum chuanxiong (LC) inhibits platelet-derived growth factor (PDGF-BB)-induced HSC proliferation. The present study was designed to investigate the active principles and their action mechanisms. With a bioactivity-directed fractionation approach, DNA synthesis (bromodeoxyuridine (BrdU) incorporation), cell cycle related proteins and apoptosis markers were determined to evaluate the inhibitory effects of active principles of LC. Two phthalides, Z,Z'-6,8',7,3'-diligustilide (1) and levistolide A (2), from LC significantly abrogated PDGF-BB-induced proliferation in both rat and human HSC lines. These inhibitory effects of compounds 1 and 2 were associated with reduction of alpha-smooth muscle actin and collagen expressions. The cell cycle promoting proteins, cyclins D1, D2, E, A and B1, were downregulated while the inhibitory proteins p21 and 27 were up-regulated. JNK phosphorylation was up-regulated by compounds 1 and 2. In HSC-T6, the two compounds induced apoptosis through the activation of caspases 9 and 3, increase in cytosolic cytochrome c release, and downregulation of Bcl-2 and Akt phosphorylation. Moreover, neither phthalides caused direct cytotoxicity to either HSCs or rat primary hepatocytes under experimental concentrations. These results indicate that two phthalides from LC inhibited PDGF-BB-activated HSC proliferation possibly through cell cycle inhibition and apoptosis mechanisms. They might be potential anti-fibrotic drugs for the treatment and prevention of hepatic fibrosis.