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CBLC (CBL proto-oncogene c) belongs to the CBL protein family, which has E3 ubiquitin ligase activity toward activated receptor tyrosine kinases. CBLC is frequently upregulated in non–small cell lung cancer (NSCLC), yet very little is known about the functions of CBLC in tumorigenesis. Here we show that CBLC is an epigenetically demethylated target and its expression can be upregulated in NSCLC after treatment with the DNA methylation inhibitor 5′-azacytidine. Depletion of CBLC significantly inhibited cell viability and clonogenicity in vitro and reduced tumor growth in a xenograft model. CBLC silencing further sensitized EGFR-mutated NSCLC cells to treatment with tyrosine kinase inhibitors. Conversely, ectopic expression of CBLC enhanced the activation of EGFR and downstream ERK1/2 signaling after ligand stimulation by competing with CBL for EGFR binding. Analysis of ubiquitin linkages on activated EGFR (aEGFR) revealed that CBLC ubiquitinated and positively regulated aEGFR stability through the conjugation of polyubiquitin by K6 and K11 linkages. This CBLC-mediated polyubiquitination promoted either preferential recycling of aEGFR back to the plasma membrane or trafficking to the cell nucleus. IHC analyses revealed a positive correlation between phospho-EGFR and CBLC in lung adenocarcinoma. In summary, we demonstrate a novel mechanism by which aEGFR escapes lysosomal degradation in a CBLC/ubiquitin-dependent manner to sustain its activation. Our work identifies CBLC as a potential diagnostic biomarker and also points to its utilization as a novel therapeutic target for NSCLC therapy.Significance: This work demonstrates the role of CBLC expression as a diagnostic biomarker and potential therapeutic target in lung adenocarcinoma. Cancer Res; 78(17); 4984–96. ©2018 AACR.

Cell differentiation within stem cell lineages can check proliferative potential, but nodal pathways that can limit tumor growth are obscure. Here, we report that lung cancer cell populations generate phenotypic and oncogenic plasticity via a switch between differentiation programs controlled by SOX2 and SOX9, thus altering proliferative and invasive capabilities. In lung cancer cells, SOX2 bound the EPCAM promoter to induce EpCAM–p21Cip1–cyclin A2 signaling, encouraging cell proliferation as well as barrier properties. In contrast, SOX9 bound the SLUG promoter to induce SLUG-mediated cell invasion with a spindle-like phenotype. Pharmacologic inhibition of HDAC elevated a SOX9-positive cell population from SOX2-positive cells, whereas ectopic expression of SOX2 inhibited SOX9 with increased H3K9me2 levels on the SOX9 promoter. In clinical specimens, the expression of SOX2 and SOX9 correlated negatively and positively with lung tumor grade, respectively. Our findings identify SOX2 and SOX9 as nodal epigenetic regulators in determining cancer cell plasticity and metastatic progression. Cancer Res; 76(23); 7036–48. ©2016 AACR.

This file contains figure legends.

This file contains technical details of experimental methods used in this study: 1. Colony formation assay; 2. Matrigel invasion assay; 3. Live cell migration assay; 4. Flow cytometry analysis; 5. Immunostaining; 6. ChIP-qPCR; 7. Cell proliferation assay; 8. Animal experiment.

Figure S1- CBLC expression is upregulated in NSCLC cell lines and clinical patients. Figure S2- Negative correlation between CBLC promoter methylation and mRNA expression in TCGA LUAD and LUSC studies.

This file contains following figures and tables: Fig. S1. Differential effects of cisplatin on SOX2- and SOX9-positive lung cancer cells (related to Fig. 1); Fig. S2. STR profiling in the derived stable clones (related to Fig. 2); Fig. S3. Expression switching between SOX2 and SOX9 (related to Fig. 2 and 3); Fig. S4. Ectopic expression of SOX2 attenuates SOX9 expression (related to Fig. 3); Fig. S5. Differential effects of SOX2 and SOX9 on cell proliferation (related to Fig. 3 and 4); Fig. S6. CCNA2-silencing attenuates cell growth in CL1-0 cells (related to Fig. 3); Fig. S7. SOX9-silencing attenuates lung invasion (related to Fig. 5); Fig. S8. Switching enrichment of H3K9me2 between CL1-1 and CL1-2 cells (related to Fig. 6); Fig. S9. Correlation analysis of SOX2 with SOX9, CCNA2 and HDAC1 in primary lung adenocarcinoma (related to Fig. 7); Fig. S10. Model for cancer cell plasticity (related to Fig. 1 to 7); Table S1. shRNA clones used in this study; Table S2. Primary antibodies used in this study; Table S3. Primers and probes used in this study; Table S4. Gene expression profiling data from the public domain used in this study.

MicroRNAs (miRNA) mediate distinct gene regulatory pathways triggered by epidermal growth factor receptor (EGFR) activation, which occurs commonly in lung cancers with poor prognosis. In this study, we report the discovery and mechanistic characterization of the miRNA miR-7 as an oncogenic “oncomiR” and its role as a key mediator of EGFR signaling in lung cancer cells. EGFR activation or ectopic expression of Ras as well as c-Myc stimulated miR-7 expression in an extracellular signal-regulated kinase (ERK)–dependent manner, suggesting that EGFR induces miR-7 expression through a Ras/ERK/Myc pathway. In support of this likelihood, c-Myc bound to the miR-7 promoter and enhanced its activity. Ectopic miR-7 promoted cell growth and tumor formation in lung cancer cells, significantly increasing the mortality of nude mice hosts, which were orthotopically implanted with lung cancers. Quantitative proteomic analysis revealed that miR-7 decreased levels of the Ets2 transcriptional repression factor ERF, the coding sequence of which was found to contain a miR-7 complementary sequence. Indeed, ectopic miR-7 inhibited production of ERF messages with a wild-type but not a silently mutated coding sequence, and ectopic miR-7 rescued growth arrest produced by wild-type but not mutated ERF. Together, these results identified that ERF is a direct target of miR-7 in lung cancer. Our findings suggest that miR-7 may act as an important modulator of EGFR-mediated oncogenesis, with potential applications as a novel prognostic biomarker and therapeutic target in lung cancer. Cancer Res; 70(21); 8822–31. ©2010 AACR.

Supplementary Methods, Figures 1-6, Tables 1-3 from EGFR Promotes Lung Tumorigenesis by Activating miR-7 through a Ras/ERK/Myc Pathway That Targets the Ets2 Transcriptional Repressor ERF

Supplementary Table 4 from EGFR Promotes Lung Tumorigenesis by Activating miR-7 through a Ras/ERK/Myc Pathway That Targets the Ets2 Transcriptional Repressor ERF

CBL family proteins (CBL, CBLB and CBLC in mammals) are E3 ubiquitin ligases of protein tyrosine kinases. CBL mediates the lysosomal degradation of activated EGFR through K63-linked ubiquitination, while CBLC has an oncogenic function by positively regulating EGFR activation through K6 and K11-linked ubiquitination in EGFR mutant lung adenocarcinoma (LAD). Here, we used immunoprecipitation and mass spectrometry to study the CBLC interactome, and found that CBLC is also involved in cell cycle regulation by stabilizing Aurora kinase A (AURKA). CBLC interacted with the kinase domain of AURKA and positively regulated the stability of AURKA by conjugating monoubiquitination and K11/K63-linked polyubiquitination, which are protective from degrading K11/K48 polyubiquitination. CBLC depletion markedly decreased the half-life of AURKA in cycloheximide-treated LAD cells. When LAD cells were synchronized with double thymidine block at the G1/S boundary and then released into mitotic arrest, CBLC depletion delayed the accumulation and activation of AURKA and prevented cancer cells from entering mitosis. CBLC deficiency significantly delayed cell cycle progression, reduced the mitotic population, and increased apoptosis of LAD cells. Targeting CBLC inhibited tumor growth of LAD cells and enhanced their sensitivity to paclitaxel in xenograft models. Immunohistochemical staining of the tissue microarray also revealed a positive correlation between the expression of CBLC and AURKA in normal and LAD tissues, further supporting the positive regulation of AURKA expression by CBLC. In summary, these findings indicate that the oncogenic E3 ligase CBLC plays a role in mitotic entry by stabilizing AURKA via ubiquitination in LAD. This work demonstrates that targeting CBLC combined with paclitaxel might be a potential option for the treatment of LAD patients who have no available targeted therapies.

Epithelial growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been widely used in the clinical treatment of non-small cell lung cancer (NSCLC) patients with EGFR mutations. Previous studies have shown that Aurora kinase A (AURKA) is overexpressed in a broad spectrum of cancer cells, which can induce epithelial-mesenchymal transition (EMT) and contribute to the occurrence of acquired EGFR-TKI resistance. However, whether the inhibition of AURKA could overcome EGFR-TKI resistance or reverse the EMT in TKI-resistant NSCLC cells remains unclear. In the current study, we established three EGFR-TKI-resistant cell lines and analyzed their expression profiles by RNA sequencing. The results revealed that the EMT pathway is significantly upregulated in the three cell lines with EGFR-TKI resistance. The phosphorylation of AURKA at Thr 288 was also upregulated, suggesting that the activation of AURKA plays an important role in the occurrence of EGFR-TKI resistance. Interestingly, the AURKA inhibitor, alisertib treatment restored the susceptibility of resistant cells to EGFR-TKIs and partially reversed the EMT process, thereby reducing migration and invasion in EGFR-TKI-resistant cells. This study provides evidence that targeting AURKA signaling pathway by alisertib may be a novel approach for overcoming EGFR-TKI resistance and for the treatment of metastatic EGFR-TKIs in NSCLC patients.

// Shih-Hsin Hsiao 1, 2 , Yu-Ting Chou 3 , Sey-En Lin 4 , Ru-Chun Hsu 2 , Chi-Li Chung 2, 5 , Yu-Rung Kao 6 , H. Eugene Liu 7, 8 and Cheng-Wen Wu 1, 3, 6, 9 1 Program in Molecular Medicine, School of Life Sciences, National Yang-Ming University and Academia Sinica, Taipei 112, Taiwan 2 Division of Pulmonary Medicine, Department of Internal Medicine, Taipei Medical University Hospital, Tapei 110, Taiwan 3 Institute of Biotechnology, College of Life Science, National Tsing Hua University, Hsinchu 30013, Taiwan 4 Department of Pathology, Wang Fang Hospital, Taipei Medical University, Tapei 11696, Taiwan 5 Division of Thoracic Medicine, Department of Internal Medicine, School of Medicine and School of Respiratory Therapy, College of Medicine, Taipei Medical University, Taipei 110, Taiwan 6 Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan 7 Division of Hematology and Oncology, Department of Internal Medicine, Wan Fang Hospital, Taipei Medical University, Tapei 11696, Taiwan 8 Graduate Institute of Clinical Medicine, Collage of Medicine, Taipei Medical University, Tapei 110, Taiwan 9 Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei 112, Taiwan Correspondence to: Cheng-Wen Wu, email: cwwu@ym.edu.tw Keywords: EGFR mutation, EGFR exon 19 deletion or L858R point mutation, non-small cell lung cancer, brain metastases Received: February 18, 2017 Accepted: May 21, 2017 Published: June 16, 2017 ABSTRACT Non-small cell lung cancer (NSCLC) patients tend to develop brain metastases (BM), but the link between BM occurrence and driver mutations in NSCLC is not very clear. We explored whether activating mutations of epidermal growth factor receptors ( EGFRs ) in exon 19 deletion or L858R predict BM in NSCLC. A retrospective multivariable logistic regression analysis of 384 patients demonstrated that the presence of mutated- EGFRs was associated with overall BM (OR=2.24, P=0.001) compared to that of wild-type EGFR (WT- EGFR ). Moreover, the time-to-event analysis model considering death as a competing risk revealed that, irrespective of survival, mutated- EGFR s predicted subsequent BM (SBM) in stage IIIB-IV patients (37.1% vs. 10.6%, HR=2.98, P=0.002) after adjusting for age (HR=2.00, P=0.012), gender, histological subtype, and smoking history. Notably, the younger mutated- EGFR subgroup was at a higher risk for SBM compared to the older WT- EGFR one (58.1% vs. 10.9%, HR=6.57, P

// Erh-Hsuan Lin 1, 2, 3, 4 , Yu-Rung Kao 1 , Chih-An Lin 5 , Ting-Yu Kuo 4 , Sheng-Ping Yang 2 , Chiung-Fang Hsu 1 , Teh-Ying Chou 3, 6 , Chao-Chi Ho 7 , Cheng-Wen Wu 1, 2, 3, 4 1 Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 2 Institute of Microbiology and Immunology, National Yang Ming University, Taipei, Taiwan 3 Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan 4 Institute of Biochemistry and Molecular Biology, National Yang Ming University, Taipei, Taiwan 5 Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University Medical College, Taipei, Taiwan 6 Department of Pathology and Laboratory Medicine, Taipei Veterans General Hospital, Taipei, Taiwan 7 Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University Medical College, Taipei, Taiwan Correspondence to: Cheng-Wen Wu, e-mail: cwwu@ym.edu.tw Keywords: lung adenocarcinoma, Hedgehog pathway, HHIP, drug resistance, HGF/MET Received: July 30, 2015 Accepted: March 02, 2016 Published: March 22, 2016 ABSTRACT Hedgehog (HH) pathway plays an important role in embryonic development, but is largely inactive in adult except for tissue repair. Aberrant activation of HH pathway has been found in a variety of cancer types. In non-small cell lung cancer, however, the role and importance of HH pathway remain controversial. In the current study, we found that HH pathway was maintained in low activity in lung adenocarcinoma (LAC) cells under normal culture condition, but was highly induced in response to stress conditions. Activation of HH pathway promoted cell survival, growth, and invasion partially through HGF and MET signaling. Hedgehog-Interacting Protein (HHIP), a cell-surface negative regulator of HH pathway, was epigenetically silenced in LAC. Overexpression of HHIP blocked the activation of HH and HGF/MET pathways, and made cells significantly more susceptible to stress conditions. In LAC cells with acquired resistance to Epidermal Growth Factor Receptor Tyrosin Kinase Inhibitor (EGFR-TKI), we found that a part of tumor cells were much more sensitive to HH or HGF/MET inhibitors, suggesting an oncogenic addiction shift from EGFR to HH and HGF/MET pathways. In conclusion, this study showed that HH pathway is a survival signaling that drives LAC cell growth under stress conditions, and HHIP is a key regulator to block the induction of HH pathway. Targeting the HH pathway through inhibitors or HHIP thus holds promise to address EGFR-TKI resistance in LAC in clinic.

Patients with lung adenocarcinoma harboring driver genes can be treated with target therapies, such as tyrosine kinase inhibitors for epithelial growth factor receptor (EGFR) mutations, anaplastic lymphoma kinase (ALK) inhibitors for ALK gene rearrangements, and so on. However, a large portion of lung adenocarcinoma patients, especially for those with wild-type EGFR, still do not have valid target therapies in clinics. The CBL family proteins (CBL, CBLB, and CBLC) are E3 ubiquitin ligases of protein tyrosine kinases (PTKs), including EGFR, hepatocyte growth factor receptor (MET), and SRC proto-oncogene (SRC). It has been reported that the K63-linkaged ubiquitination mediated by CBL can negatively regulate activated EGFR (aEGFR) signaling through lysosomal degradation. Previously, we discovered that CBLC, unlike CBL, possesses an oncogenic function and positively regulates aEGFR stability through polyubiquitination via K6 and K11 linkages. The CBLC-mediated conjugation of polyubiquitin promotes aEGFR preferentially recycled back to the plasma membrane, or trafficked to the cell nucleus, leading to the sustained activation of EGFR signaling in lung adenocarcinoma. Aside from aEGFR dysregulation, we recently found that CBLC assumes a novel role in positively regulating the expression of Aurora kinase A (AURKA), which contributes to tumor development by promoting cell proliferation, metastasis, epithelial-mesenchymal transition, and drug resistance. In lung adenocarcinoma cells with wild-type EGFR, CBLC knockdown significantly reduced cell viability, delayed cell cycle progression and increased apoptotic death. Our study also showed that CBLC knockdown in EGFR wild type cells was associated with increased sensitivity to paclitaxel, probably through downregulating AURKA. Taken together, these findings indicated that CBLC may play a role in promoting cell cycle progression and may be a novel therapeutic target to enhance the efficacy of paclitaxel on EGFR wild-type lung adenocarcinoma. Citation Format: Shiao-Ya Hong, Yu-Rung Kao, Yi-Ping Lin, Meng-Hsuan Lee, Jyun-Yi Wu, Cheng-Wen Wu. CBLC functions as a novel therapeutic target to enhance the efficacy of paclitaxel on EGFR wild-type lung adenocarcinoma by downregulating AURKA [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 4852.

CBLC (CBL proto-oncogene c) belongs to the CBL protein family, which has E3 ubiquitin ligase activity toward activated receptor tyrosine kinases. CBLC is frequently upregulated in non–small cell lung cancer (NSCLC), yet very little is known about the functions of CBLC in tumorigenesis. Here we show that CBLC is an epigenetically demethylated target and its expression can be upregulated in NSCLC after treatment with the DNA methylation inhibitor 5′-azacytidine. Depletion of CBLC significantly inhibited cell viability and clonogenicity in vitro and reduced tumor growth in a xenograft model. CBLC silencing further sensitized EGFR-mutated NSCLC cells to treatment with tyrosine kinase inhibitors. Conversely, ectopic expression of CBLC enhanced the activation of EGFR and downstream ERK1/2 signaling after ligand stimulation by competing with CBL for EGFR binding. Analysis of ubiquitin linkages on activated EGFR (aEGFR) revealed that CBLC ubiquitinated and positively regulated aEGFR stability through the conjugation of polyubiquitin by K6 and K11 linkages. This CBLC-mediated polyubiquitination promoted either preferential recycling of aEGFR back to the plasma membrane or trafficking to the cell nucleus. IHC analyses revealed a positive correlation between phospho-EGFR and CBLC in lung adenocarcinoma. In summary, we demonstrate a novel mechanism by which aEGFR escapes lysosomal degradation in a CBLC/ubiquitin-dependent manner to sustain its activation. Our work identifies CBLC as a potential diagnostic biomarker and also points to its utilization as a novel therapeutic target for NSCLC therapy. Significance: This work demonstrates the role of CBLC expression as a diagnostic biomarker and potential therapeutic target in lung adenocarcinoma. Cancer Res; 78(17); 4984–96. ©2018 AACR.

The epithelial growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been widely used to treat EGFR-mutant non-small cell lung cancer (NSCLC) in clinics. Several generations of TKI were invented to overcome the unfavorable response and acquired resistance after long-term usage of EGFR TKI. Several mechanisms of acquired resistance have been reported. The well-studied resistant mechanism results from EGFR secondary mutations including T790M and C797S, which are involved in the resistance to first and third generation TKI, respectively. Other mechanisms aside from secondary mutation on EGFR including Her2 and MET amplification or epithelial mesenchymal transition (EMT) also distribute to TKI resistance. However, the resistant mechanisms which are independent to these alterations have not been completely identified yet. Here, we generated resistant cells selected by three different generations of TKI and analyzed their gene expression profiles. We discovered that many signal pathways, especially involved in cell cycle regulation, are commonly upregulated in three TKI-resistant cell lines. Aurora kinase A (AURKA) was the upstream kinase of those pathways; however, neither RNA level nor protein level of AURKA was significant changed. Interestingly, we found that phosphorylation of Aurora A at Thr288 in its catalytic domain is higher in TKI resistant cells. This reveals the possibilities that AURKA activation plays a role in the occurrence of TKI-resistance and that TKI-resistant NSCLC patients have a better response rate to AURKA inhibitor treatments. Citation Format: Meng-Hsuan Lee, Shiao-Ya Hong, Yu-Rung Kao, Cheng-Wen Wu. Activation of AURKA contributes to TKI resistance in EGFR-mutant non-small lung cancer [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 4756.

// 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.

MicroRNAs (miRNA) mediate distinct gene regulatory pathways triggered by epidermal growth factor receptor (EGFR) activation, which occurs commonly in lung cancers with poor prognosis. In this study, we report the discovery and mechanistic characterization of the miRNA miR-7 as an oncogenic “oncomiR” and its role as a key mediator of EGFR signaling in lung cancer cells. EGFR activation or ectopic expression of Ras as well as c-Myc stimulated miR-7 expression in an extracellular signal-regulated kinase (ERK)–dependent manner, suggesting that EGFR induces miR-7 expression through a Ras/ERK/Myc pathway. In support of this likelihood, c-Myc bound to the miR-7 promoter and enhanced its activity. Ectopic miR-7 promoted cell growth and tumor formation in lung cancer cells, significantly increasing the mortality of nude mice hosts, which were orthotopically implanted with lung cancers. Quantitative proteomic analysis revealed that miR-7 decreased levels of the Ets2 transcriptional repression factor ERF, the coding sequence of which was found to contain a miR-7 complementary sequence. Indeed, ectopic miR-7 inhibited production of ERF messages with a wild-type but not a silently mutated coding sequence, and ectopic miR-7 rescued growth arrest produced by wild-type but not mutated ERF. Together, these results identified that ERF is a direct target of miR-7 in lung cancer. Our findings suggest that miR-7 may act as an important modulator of EGFR-mediated oncogenesis, with potential applications as a novel prognostic biomarker and therapeutic target in lung cancer. Cancer Res; 70(21); 8822–31. ©2010 AACR.

CBLC (CBL proto-oncogene c) belongs to the CBL protein family which has E3 ubiquitin ligase activity towards activated receptor tyrosine kinases. CBLC expression is physiologically restricted to normal epithelial cells but frequently upregulated in non-small cell lung cancer (NSCLC). Compared to its family members, very little is known about the functions of CBLC in tumorigenesis. Here, we demonstrated that CBLC is an epigenetically demethylated target and its expression can be upregulated after treatment with a DNA methylation inhibitor, 5'-azacytidine in NSCLC cells. CBLC depletion significantly inhibited cell viability and clonogenicity in vitro and reduced the tumor growth in a xenograft model. In addition, CBLC silencing further sensitized EGFR-mutated NSCLC cells to tyrosine kinase inhibitors treatment. On the other hand, after ligand stimulation, ectopic expression of CBLC enhanced the activation of EGFR and its downstream ERK1/2 signaling via competing with CBL for EGFR binding. By analyzing the ubiquitin linkages on activated EGFR (aEGFR), we found that CBLC ubiquitinated and positively regulated aEGFR stability through the conjugation of polyubiquitin by K6 and K11 linkages. The CBLC-mediated polyubiquitination promoted aEGFR preferentially recycling back to the plasma membrane or trafficking to the cell nucleus. In summary, we demonstrate a novel mechanism by which aEGFR escape lysosomal degradation in a CBLC/ubiquitin-dependent manner to sustain its activation. Our work identifies CBLC as a potential diagnostic biomarker and also points to its utilization as a novel therapeutic target for NSCLC therapy. Citation Format: Shiao-Ya Hong, Yu-Rung Kao, Te-Chang Lee, Cheng-Wen Wu. Epigenetic upregulation of E3 ubiquitin ligase CBLC enhances EGFR dysregulation and signaling [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 3319.

CBL family proteins have been reported as E3 ubiquitin ligases of receptor tyrosine kinases. CBLC is the most divergent member of CBL family in human due to lack of the distal part of C terminus found in other two proteins, CBL and CBLB. By data mining from TCGA database, CBLC expression was frequently upregulated in lung adenocarcinoma. However, very little is known about the tumorigenic functions of CBLC compared to other CBL family proteins. Although patients with lung adenocarcinoma harboring EGFR mutations can be treated with tyrosine kinase inhibitors (TKIs), there is no good target therapy for treating lung adenocarcinoma patients with EGFR wild-type or TKIs resistance. Here, this study is to investigate if CBLC can be a good target for treatment to benefit these patients. In lung adenocarcinoma cells, CBLC knockdown significantly decreased cell viability, while CBLC overexpression increased the ability to form colonies in soft agar. Flow cytometry analyses revealed that CBLC depletion decreased the cell population of G2/M phase and increased cell apoptosis. Our study also showed that CBLC knockdown in EGFR wild-type cells increased the drug resistance to paclitaxel, which is widely used as a first-line chemotherapy drug in lung adenocarcinoma patients with EGFR wild-type or TKI resistance. Taken together, these findings indicate that CBLC might play a role in promoting cell mitosis and could be a potential biomarker of paclitaxel susceptibility for patients with EGFR wild-type lung adenocarcinoma. Citation Format: Yi-Ping Lin, Shiao-Ya Hong, Yu-Rung Kao, Cheng-Wen Wu. CBLC is a potential biomarker for the susceptibility to paclitaxel 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 2642.

Cell differentiation within stem cell lineages can check proliferative potential, but nodal pathways that can limit tumor growth are obscure. Here, we report that lung cancer cell populations generate phenotypic and oncogenic plasticity via a switch between differentiation programs controlled by SOX2 and SOX9, thus altering proliferative and invasive capabilities. In lung cancer cells, SOX2 bound the EPCAM promoter to induce EpCAM–p21Cip1–cyclin A2 signaling, encouraging cell proliferation as well as barrier properties. In contrast, SOX9 bound the SLUG promoter to induce SLUG-mediated cell invasion with a spindle-like phenotype. Pharmacologic inhibition of HDAC elevated a SOX9-positive cell population from SOX2-positive cells, whereas ectopic expression of SOX2 inhibited SOX9 with increased H3K9me2 levels on the SOX9 promoter. In clinical specimens, the expression of SOX2 and SOX9 correlated negatively and positively with lung tumor grade, respectively. Our findings identify SOX2 and SOX9 as nodal epigenetic regulators in determining cancer cell plasticity and metastatic progression. Cancer Res; 76(23); 7036–48. ©2016 AACR.

Cell invasion and angiogenesis are crucial processes in cancer metastasis that require extracellular matrix (ECM) degradation. Proteolytic degradation of the ECM components is a central event of invasion and angiogenesis processes. During these processes, matrix metalloproteinases (MMPs) seem to be primarily responsible for much of the ECM degradation. Disulfiram is frequently used in the treatment of alcoholism and has been reported to possess antiretroviral activity and can eject intrinsic zinc out of human immunodeficiency virus (HIV) nucleocapsid protein. In this report, we show that disulfiram inhibited invasion and angiogenesis in both tumor and endothelial cells at nontoxic concentrations. The 3H-labeled type IV collagen degradation assay suggested that disulfiram has type IV collagenase inhibitory activity, and this inhibition was responsible for blocking invasion and angiogenesis through cell-mediated and non-cell-mediated pathways. However, the mechanisms underlying cell-mediated signal pathways are not fully characterized. Our data demonstrate that the non-cell-mediated pathway is dominant. Thus, disulfiram could directly interact with MMP-2 and MMP-9 and inhibit their proteolytic activity through a zincchelating mechanism. Addition of zinc could reverse the inhibition of invasiveness and collagenase inhibition through disulfiram treatment. This finding implies that MMP-2 and MMP-9 may be the inhibitory targets for a potential disulfiram treatment. These observations raise the possibility clinical therapeutic applications for disulfiram used as a potential inhibitor of metastatic cell invasion and angiogenesis.

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.

Aberrant expression and function of epidermal growth factor receptor (EGFR) are reported in most lung cancer cases and are correlated with poor prognosis. However, the role of many downstream effectors associated with abnormal EGFR signaling is not clear in cancer progression. Cited2, a novel transcriptional co-activator was identified to be induced by hyperactive EGFR mutant L858R in lung cancer cells. Overexpression of Cited2 in CL1-0 lung cancer cells allowed cell to survive in serum free condition. Knockdown of Cited2 attenuated cell growth, leading to G1/S cell cycle arrest. Through microarray analysis, we identified that expression of E2F3 and its downstream gene targets were blocked in Cited2-silenced cells. Overexpression of Cited2 enhanced E2F3 expression, supporting that Cited2 regulates E2F3 expression. By chromatin immunoprecipitation assay, we found Cited2 bound to E2F3 promoter. In addition to affecting cell growth, knockdown of Cited2 promoted cell senescence with flat and enlarged cell morphology, and increased senescence-associated ß-galactosidase activity. Through western blot analysis, we found Cited2-silencing induced p53 and p21 expression. Overexpression of Cited2 further enhanced tumor growth in nude mice. Clinical analysis showed that Cited2 was highly expressed in lung cancer but not in normal tissues. These data demonstrate that Cited2 plays a key role in lung cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 274.