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Correlations between CEBPD expression and other important clinicopathological parameters in 79 muscle invasive UCUB receiving post-operative chemotherapy

Supplementary Figures S1-S4.

Correlations between expression status of pEGFR, pSTAT3, CEBPD, and ABCB1 in muscle invasive UCUB in our independent cohort containing 60 patients receiving post-operative chemotherapy

Attenuated pSTAT3, CEBPD, ABCB1 and ABCC2 mRNA levels are observed in CDDP-resistant NTUB1/P xenografted NOD/SCID mice. A and B, Following 3 weeks of drug treatment, all animals were sacrificed, and their tumors were collected for protein and mRNA analysis. Protein samples were employed for Western blot analysis by using specific antibodies. The following data present changes in pSTAT3, CEBPD, and ABCB1 levels in NTUB1/P-xenografted NOD/SCID mice treated with CDDP and gefitinib or with CDDP and S3I-201. qPCR assays were performed using the total RNA harvested from tumor samples. In this figure, *** denotes a significant difference (P < 0.001). B, evaluation of body weight of experimental mice used in Fig. 5. CTL: control.

CEBPD expression had significant worse MeFS in UCUB patients. A and B, Kaplan-Meier plots discloses the predictive significances of CEBPD immunoexpression for metastasis-free survival in UCUB.

Purpose: Recent evidence indicates that a tumor suppressor gene CEBPD (CCAAT/enhancer-binding protein delta) is downregulated in many cancers including cervical cancer, which provides a therapeutic potential associated with its reactivation. However, little is known for CEBPD activators and the effect of reactivation of CEBPD transcription upon anticancer drug treatment. In this study, we identified a novel CEBPD activator, 1-(2-hydroxy-5-methylphenyl)-3-phenyl-1,3-propanedione (HMDB). The purpose of this study is to characterize the mechanism of HMDB-induced CEBPD activation and its potential effect in cancer therapy.Experimental Design: Methylation-specific PCR assay, reporter assay, and chromatin immunoprecipitation (ChIP) assay were performed to dissect the signaling pathway of HMDB-induced CEBPD transcription. Furthermore, a consequence of HMDB-induced CEBPD expression was linked with E2F1 and retinoblastoma (RB), which discloses the scenario of CEBPD, E2F1, and RB bindings and transcriptional regulation on the promoters of proapoptotic genes, PPARG2 and GADD153. Finally, the anticancer effect of HMDB was examined in xenograft mice.Results: We demonstrate that CEBPD plays an essential role in HMDB-mediated apoptosis of cancer cells. HMDB up-regulates CEBPD transcription through the p38/CREB pathway, thus leading to transcriptional activation of PPARG2 and GADD153. Furthermore, increased level of CEBPD attenuates E2F1-induced cancer cell proliferation and partially rescues RB/E2F1-mediated repression of PPARG2 and GADD153 transcription. Moreover, HMDB treatment attenuates the growth of A431 xenografts in severe combined immunodeficient mice mice.Conclusions: These results clearly demonstrate that HMDB kills cancer cells through activation of CEBPD pathways and suggest that HMDB can serve as a superior chemotherapeutic agent with limited potential for adverse side effects. Clin Cancer Res; 16(23); 5770–80. ©2010 AACR.

Levels of CEBPD associates with STAT3 activity in gefitinib-treated CDDP-resistant NTUB1/P (NTP) cells. A, Western blot analysis was conducted with lysates from NTP cells treated with gefitinib following various time courses and blotted with specific antibodies. B, gefitinib and S3I-201 attenuate expression of phosphorylation of STAT3 (pY705) and CEBPD in J82 cells. Cells were treated with CDDP alone or in combination with gefitinib or S3I-201 for 24 h. Western blot was conducted with indicated antibodies, respectively.

Expression and activities of EGFR and STAT3 levels in UCUB cells. Western blot analysis was conducted with lysates from UCUB cells. TSGH8301 (TSG), TCCSUP (TCC), NTUB1 (NTU) and CDDP resistance NTUB1/P (NTP).

Overexpression of CEBPD upregulates ABCB1 and ABCC2 transcription in CDDP-resistant NTUB1/P (NTP) cells. A, RT-PCR assay was conducted with total RNA harvested from NTP cells transfected with pCDNA3/HA/CEBPD expression vectors. B, overexpression of CEBPD upregulates ABCB1 transcription containing known exon 1 (NM_000927.4) in CDDP-resistant NTP cells. RT-PCR assay was conducted with total RNA harvested from NTP cells transfected with pCDNA3/HA/CEBPD expression vectors. Upper panel shows location of forward primer (+159) in exon 1 and reverse primer (+348) in exon 2 of ABCB1 gene locus.

Gefitinib and S3I-201 attenuate ABCB1 transcripts of and enhance CDDP sensitivity of J82 cells. A, cells were treated with CDDP alone or in combination with gefitinib or S3I-201 for 24 h. A qPCR assay was conducted with specific primers for ABCB1 gene. ***, significant difference (P < 0.001). B, J82 cells were then transfected with ABCB1 reporter (-21~+917) (AB-I, as shown in Fig. 3D) and treated with CDDP alone or in combination with gefitinib or S3I-201. After 24 h. A Luciferase assay was conducted using lysates of transfectants. ***, significant difference (P < 0.001). C, gefitinib and S3I-201 attenuate ABCB1 pump activity and enhance CDDP sensitivity of J82 cells. J82 cells were treated with CDDP alone or CDDP combining gefitinib or S3I-201 for 24 h, MDR dye-loading solution was added to each well for 4 h, and fluorescence intensities were detected by ELISA reader. ***, significant difference (P < 0.001). D, J82 cells were treated with CDDP alone or CDDP combining gefitinib or S3I-201 for 24 h. Death of experimental cells was examined by PI staining. ***, significant difference (P < 0.001). E, CEBPD contributes to drug resistance of cisplatin (CDDP) in nasopharyngeal carcinoma HONE1/R (CDDP-resistant HONE) cells. HONE1/R cells were resistant to CDDP treatment. HONE1 and HONE1/R cells were treated with CDDP in dose-dependent manner. Cell viability was examined by CCK8 assay. *, significant difference (P < 0.05). **, significant difference (P < 0.01). ***, significant difference (P < 0.001). F, CDDP induces expression of CEBPD and ABCB1. HONE1/R cells were treated with CDDP for 24 h and then lysates of experimental cells were analyzed by Western blot. G, HONE1/R cells were treated with gefitinib, S3I-201 or CDDP or in combination as indicated for 24 h, MDR dye-loading solution was added to each well for 4 h and fluorescence intensities were detected by ELISA reader. ***, significant difference (P < 0.001). H, S3I-201 enhances sensitivity of CDDP in HONE1/R cells. HONE1/R cells were treated with CDDP (20 μM) alone or in combination with S3I-201 (100 μM) for 24 h and death of experimental cells was examined by PI staining. ***, significant difference (P < 0.001).

CEBPD expression is correlated with pEGFR, pSTAT3 and ABCB1 expression in UCUB patients. Immunostaining on representative case developed metastatic disease (Case-45, Left panel) exhibits high pEGFR, pSTAT3, CEBPD, and ABCB1 expression; while the case with superior outcome (Case-32, Right panel) shows low expression of these proteins.

Purpose: Cisplatin (CDDP) is frequently used in combination chemotherapy with paclitaxel for treating urothelial carcinoma of the urinary bladder (UCUB). CDDP cross-resistance has been suggested to develop with paclitaxel, thus hindering successful UCUB treatment. Therefore, elucidating the mechanisms underlying CDDP-induced anticancer drug resistance is imperative and may provide an insight in developing novel therapeutic strategy.Experimental Design: Loss-of-function assays were performed to elucidate the role of the EGFR and STAT3 in CDDP-induced CCAAT/enhancer-binding protein delta (CEBPD) expression in UCUB cells. Reporter and in vivo DNA-binding assays were employed to determine whether CEBPD directly regulates ATP binding cassette subfamily B member 1 (ABCB1) and ATP binding cassette subfamily C member 2 (ABCC2) activation. Finally, a xenograft animal assay was used to examine the abilities of gefitinib and S3I-201 (a STAT3 inhibitor) to reverse CDDP and paclitaxel sensitivity.Results: CEBPD expression was maintained in postoperative chemotherapy patients, and this expression was induced by CDDP even in CDDP-resistant UCUB cells. Upon CDDP treatment, CEBPD activated ABCB1 and ABCC2. Furthermore, the EGFR/STAT3 pathway contributed to CDDP-induced CEBPD expression in UCUB cells. Gefitinib and S3I-201 treatment significantly reduced the expression of CEBPD and enhanced the sensitivity of CDDP-resistant UCUB cells to CDDP and paclitaxel.Conclusions: Our results revealed the risk of CEBPD activation in CDDP-resistant UCUB cells and suggested a therapeutic strategy for patients with UCUB or UCUB resisted to CDDP and paclitaxel by combination with either gefitinib or S3I-201. Clin Cancer Res; 23(2); 503–13. ©2016 AACR.

Supplementary Figure 4 from ZBRK1 Acts as a Metastatic Suppressor by Directly Regulating MMP9 in Cervical Cancer

Supplementary Figure 1 from ZBRK1 Acts as a Metastatic Suppressor by Directly Regulating MMP9 in Cervical Cancer

Supplementary Figure 5 from ZBRK1 Acts as a Metastatic Suppressor by Directly Regulating MMP9 in Cervical Cancer

Supplementary Figure 3 from ZBRK1 Acts as a Metastatic Suppressor by Directly Regulating MMP9 in Cervical Cancer

Supplementary Figure 2 from ZBRK1 Acts as a Metastatic Suppressor by Directly Regulating MMP9 in Cervical Cancer

The BRCA1-interacted transcriptional repressor ZBRK1 has been associated with antiangiogenesis, but direct evidence of a tumor suppressor role has been lacking. In this study, we provide evidence of such a role in cervical carcinoma. ZBRK1 levels in cervical tumor cells were significantly lower than in normal cervical epithelial cells. In HeLa cervical cancer cells, enforced expression inhibited malignant growth, invasion, and metastasis in a variety of in vitro and in vivo assays. Expression of the metalloproteinase MMP9, which is known to be an important driver of invasion and metastasis, was found to be inversely correlated with ZBRK1 in tumor tissues and a target for repression in tumor cells. Our findings suggest that ZBRK1 acts to inhibit metastasis of cervical carcinoma, perhaps by modulating MMP9 expression. Cancer Res; 70(1); 192–201

Supplementary Figure Legends 1-5 from ZBRK1 Acts as a Metastatic Suppressor by Directly Regulating MMP9 in Cervical Cancer

Supplementary Tables 1-2 from ZBRK1 Acts as a Metastatic Suppressor by Directly Regulating MMP9 in Cervical Cancer

Lung cancer is a malignant lung tumor characterized by high incidence and motility. c-Myc is a transcription factor that plays an important role in oncogenic activation to influence cellular metabolism and proliferation. Ubiquitin-specific peptidase 24 (USP24) is one of the members of USPs family to regulate protein ubiquitination. However, it is lack of evidence to uncover the role of USP24 in cancer formation. In this study, we found that cell proliferation was significantly increased by USP24 knockdown in A549 cells as accompanied by the increase of c-Myc protein. In particular, the RNA level and protein stability of c-Myc were not affected by USP24 knockdown, suggesting that USP24 affects c-Myc through regulating the translational pathway. Furthermore, c-Myc participated in USP24-knocked down-induced proliferation, not migration. To further clarify the mechanism underlying the regulation of c-Myc by USP24, we investigated whether mTOR pathway is involved in c-Myc upregulation by USP24 knockdown. Herein rapamycin, the mTOR inhibitor, prevented c-Myc upregulation caused by USP24 knockdown, suggesting that USP24 affect the protein level of c-Myc by regulating mTOR-mediated translation. We found that the downstream signaling of mTOR is activated by USP24 knockdown, including S6K and RPS6 phosphorylation. In addition, USP24 knockdown induced the binding of activated PRS6 to 5′-UTR of c-Myc, indicating that USP24 regulates c-Myc through mTOR-mediated translation. However, USP24 did not affect the phosphorylation of mTOR at Ser-2448, implying that other phosphorylation residue(s) within mTOR might be regulated by USP24. Since mTOR pathway is critical for many cancers formation, understanding the role of USP24 in regulating mTOR pathway will contribute to develop strategies to fight lung cancer. Citation Format: Jan-Jong Hung, Wen-Chang Chang. Ubiquitin-specific peptidase 24 decreases c-Myc expression to inhibit lung cancer formation [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 4436.

Colorectal cancer (CRC) is highly associated with metabolic diseases, such as obesity and diabetes. Elevated free fatty acids (FFAs) in cancer patients with metabolic disorders may be associated with cancer progression. In addition, increased levels of reactive oxygen species (ROS) are also observed in dyslipidemia which induces expression of NADPH oxidase 4 (NOX4) by FFAs in aorta, kidney, and white adipocyte in physiology condition. Although the expression of NOX4 has been also up-regulated in various cancer cell types, the correlation between up-regulation of NOX4 and dyslipidemia-regulated CRC metastasis remains unclear. Here, we found that oleic acid (OA) induced-angiopoietin-like 4 (ANGPTL4) expression was through the activation of PPARs pathways, resulting in promoting the metastasis of CRC. The recombinant protein of human ANGPTL4 rescued the OA-induced invasive ability in ANGPTL4 knockdown cells. It is worthy to note that the knockdown of ANGPTL4 not only significantly inhibited OA-induced NOX4 expression, but also reduced ROS production. In addition, the depletion of ROS by using NAC or knockdown of NOX4 inhibited OA-induced extravasation of CRC cells in vivo. OA-induced MMP1 and MMP9 expressions were also dependent on the expression of ANGPTL4 and NOX4 in CRC cells. The transcriptional activation of NOX4 gene by OA-induced ANGPTL4 was regulated by activation of c-Jun and dependent on AP-1 site of NOX4 promoter. These results reveal that OA-promoted CRC metastasis was through the activation of ANGPTL4/NOX4 axis, suggesting that ANGPTL4 and NOX4 may be potential therapeutic targets and diagnostic markers to improving outcomes for patients with CRC. Citation Format: Chih-Jie Shen, Yu-Han Liao, Jhih-Peng Tsai, Liang-Yi Hung, Wen-Chang Chang, Ben-Kuen Chen. Oleic acid-induced ANGPTL4 facilitates metastasis of human colorectal cancer via up-regulation of NOX4 expression [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 2855.

Purpose: Recent evidence indicates that a tumor suppressor gene CEBPD (CCAAT/enhancer-binding protein delta) is downregulated in many cancers including cervical cancer, which provides a therapeutic potential associated with its reactivation. However, little is known for CEBPD activators and the effect of reactivation of CEBPD transcription upon anticancer drug treatment. In this study, we identified a novel CEBPD activator, 1-(2-hydroxy-5-methylphenyl)-3-phenyl-1,3-propanedione (HMDB). The purpose of this study is to characterize the mechanism of HMDB-induced CEBPD activation and its potential effect in cancer therapy. Experimental Design: Methylation-specific PCR assay, reporter assay, and chromatin immunoprecipitation (ChIP) assay were performed to dissect the signaling pathway of HMDB-induced CEBPD transcription. Furthermore, a consequence of HMDB-induced CEBPD expression was linked with E2F1 and retinoblastoma (RB), which discloses the scenario of CEBPD, E2F1, and RB bindings and transcriptional regulation on the promoters of proapoptotic genes, PPARG2 and GADD153. Finally, the anticancer effect of HMDB was examined in xenograft mice. Results: We demonstrate that CEBPD plays an essential role in HMDB-mediated apoptosis of cancer cells. HMDB up-regulates CEBPD transcription through the p38/CREB pathway, thus leading to transcriptional activation of PPARG2 and GADD153. Furthermore, increased level of CEBPD attenuates E2F1-induced cancer cell proliferation and partially rescues RB/E2F1-mediated repression of PPARG2 and GADD153 transcription. Moreover, HMDB treatment attenuates the growth of A431 xenografts in severe combined immunodeficient mice mice. Conclusions: These results clearly demonstrate that HMDB kills cancer cells through activation of CEBPD pathways and suggest that HMDB can serve as a superior chemotherapeutic agent with limited potential for adverse side effects. Clin Cancer Res; 16(23); 5770–80. ©2010 AACR.

The prognosis of glioblastoma (GBM) is usually poor even following treatment with the first-line chemotherapeutic agent temozolomide (TMZ). One most known resistant mechanism is the presence of DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). However, compared with MGMT-mediated innate TMZ resistance, the development of acquired resistance is considered more complex with multi-factorial involvement such as the presence of cancer stem cells (CSCs). In this study, we treated the MGMT-negative GBM cells with TMZ to investigate the acquired resistance, and found that both histone deacetylases (HDACs) and Sp1 are key factors protecting GBM against TMZ. These results include the following: (1) Stemness markers were highly increased in TMZ-resistant GBMs; (2) The activity of HDACs affected the stem-like characteristics and cell survival of GBM stem cells (GSCs); (3) An HDAC1/2/6-selective inhibitor MPT0B291 increased TMZ-sensitivity and induced senescence in TMZ-resistant cells; (4) MPT0B291 suppressed anti-senescence genes (hTERT and BMI1) expression via inhibition Sp1 transactivation; (5) Both HDACs and Sp1 were elevated and interacted with each other in GSCs and resistant GBM cells; (6) TMZ treatment induced Sp1 deacetylation, but MPT0B291 attenuated that. In summary, we verified that HDACs increases Sp1 activation via protein deacetylation and causes Sp1-downstream target upregulation, which may enrich stemness properties and protect GBM against chemotherapeutic drugs. Citation Format: Jian-Ying Chuang, Che-Chia Hsu, Kwang-Yu Chang, Wen-Chang Chang. Sp1 acetylation associates with stemness characteristics in temozolomide-resistant glioblastoma [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 5905.

Obese patients have higher levels of free fatty acids (FFAs) in their plasma and a higher risk of cancer than their non-obese counterparts. However, the mechanisms involved in the regulation of cancer metastasis by FFAs remain unclear. In this study, we found that oleic acid (OA) induced angiopoietin-like 4 (ANGPTL4) protein expression and secretion and conferred anoikis resistance to head and neck squamous cell carcinomas (HNSCCs). The autocrine production of OA-induced ANGPTL4 further promoted HNSCC migration and invasion. In addition, the expression of peroxisome proliferator-activated receptor (PPAR) was essential for the OA-induced ANGPTL4 expression and invasion. The levels of OA-induced epithelial-mesenchymal transition markers, such as vimentin, MMP-9, and fibronectin and its downstream effectors Rac1/Cdc42, were significantly reduced in ANGPTL4-depleted cells. Knocking down fibronectin not only inhibited the expression of MMP-9 but also repressed OA- and recombinant ANGPTL4-induced HNSCC invasion. On the other hand, ANGPTL4 siRNA inhibited OA-induced MMP-9 expression, which was reversed in fibronectin-overexpressing cells. Furthermore, the depletion of ANGPTL4 impeded the OA-primed metastatic seeding of tumor cells in the lungs. These results demonstrate that OA enhances HNSCC metastasis through the ANGPTL4/fibronectin/Rac1/Cdc42 and ANGPTL4/fibronectin/MMP-9 signaling axes. The inhibition of ANGPTL4 could be a potential strategy for the treatment of FFA-mediated HNSCC metastasis. Citation Format: Chih-Jie Shen, Shih-Hung Chan, Chung-Ta Lee, Wan-Chen Huang, Jhih-Peng Tsai, Kwang-Yu Chang, Wen-Chang Chang, Ben-Kuen Chen. Oleic acid promotes head and neck squamous cell carcinoma anoikis resistance and metastasis via ANGPTL4/fibronectin pathway [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 85.

The resisitant mechanism of glioblastoma mutliforme (GBM) to the standard chemotherapy temozolomide (TMZ) can be attributed to methylguanine methyltransferase (MGMT) expression in only half of the clinical cases. For the other half, it is not fully understood. To elucidate the mechanism, MGMT-negative GBM cell lines, U87MG and A172, were used to develop the TMZ-resistant variants. The variants showed reduced reactive oxygen species (ROS) accumulation by TMZ treatment comparing to the parental one. Further analysis of the cells revealed enhanced expression of superoxide dismutases 2 (SOD2), an antioxidative enzyme, as well as the stem cell-like properties. The protein was shown to associate with glioma stem cells (GSC). Notably, inhibition of SOD2 by diethyldithiocarbamate attenuated the stemness properties and the viability of the resistant variants. Further investigation of the promotor binding study suggested specificity protein 1 (Sp1) as the key factor of the SOD2 response. Moreover, Sp1 expression was even enhanced in the spheroid cells and the TMZ-resistant U87MG cell line. Conversely, treatment with Sp1 inhibitor mithramycin A attenuates SOD2 expression and the stemness properties of the resistant variants. In summary, our results suggested a novel role of SOD2 in TMZ resistant mechanism of GBM. Further study of Sp1-SOD2 pathway as a therapeutic target to restore susceptibility of chemotherapy is therefore warranted. Citation Format: Jian-Ying Chuang, Jr-Jiun Liu, Shao-Wen Chou, Wen-Chang Chang, Kwang-Yu Chang. Upregulated superoxide dismutase 2 by specificity protein 1 mediates temozolomide resistance in glioma stem cells. [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 5037.

Epidermal growth factor receptor (EGFR) activation is a major cause of metastasis in many cancers, such as head and neck squamous cell carcinoma (HNSCC). However, whether the induction of pyruvate dehydrogenase kinase 1 (PDK1) mediates EGF-enhanced HNSCC metastasis remains unclear. Interestingly, we found that EGF induced PDK1 expression in HNSCC. The tumor cell transformation induced by EGF was repressed by the depletion of PDK1. The down-regulation of PDK1 expression or inhibition of its activity significantly blocked EGF-enhanced cell migration and invasion. In addition, depletion of PDK1 impeded EGF-enhanced binding of HNSCC cells to endothelial cells and tumor cells metastastic seeding of the lungs. Knockdown of PDK1 also inhibited EGF-induced matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-9, and fibronectin expression and Rac1/cdc42 activation. These results demonstrate that EGF-induced PDK1 expression enhances HNSCC metastasis via activation of the fibronectin signaling pathway. The inhibition of PDK1 may be a potential strategy for the treatment of EGFR-mediated HNSCC metastasis. Citation Format: Ben-Kuen Chen, Jinn-Yuan Hsu, Wen-Chang Chang. Epidermal growth factor-induced pyruvate dehydrogenase kinase 1 enhances head and neck squamous cell carcinoma metastasis through fibronectin up-regulation. [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 710.

The aryl hydrocarbon receptor nuclear translocator (ARNT) is widely involved in regulating the tumorigenesis process by inducing genes that are involved in tumor growth and angiogenesis. Tumorigenesis usually involves normoxic conditions. However, the role of ARNT in tumor metastasis during normoxia remains unclear. Here, we demonstrated that ARNT protein levels were decreased in late stage human colorectal cancer using an immunohistochemical analysis. Stably silence of the ARNT protein promoted cancer cell migration and invasion, which was mediated by the activation of the fibronectin/integrin β1/FAK signaling axis. In addition, ARNT knockdown-induced migration and invasion was inhibited when ARNT was restored in cells. In a xenograft analysis of severe combined immunodeficiency mice, ARNT-knockdown inhibited tumor growth. However, when ARNT expression recovered, the tumor growth of ARNT-knockdown-induced metastatic lung colonies was significantly enhanced. Interestingly, chemotherapeutic drugs inhibited ARNT expression and promoted invasion of residual tumor cells. These results suggested that ARNT may play a positive role during tumor growth (either in early stage tumor growth or in metastatic organs), but plays a negative role in tumor migration and invasion. Therefore, the efficiency of ARNT-targeted therapy in different cancer stages should be carefully evaluated. Citation Format: Chi-Ruei Huang, Chung-Ta Lee, Kwang-Yu Chang, Wen-Chang Chang, Ben-Kuen Chen. Decrease of ARNT promotes cancer metastasis by activating the fibronectin/integrin β1/FAK axis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 523. doi:10.1158/1538-7445.AM2015-523

Epidermal growth factor receptor (EGFR) activation is a major cause of cell metastasis in many cancers, such as head and neck squamous cell carcinoma (HNSCC). However, whether the induction of cyclooxygenase-2 (COX-2) correlates with EGF-enhanced HNSCC metastasis remains unclear. Interestingly, we found that EGF induced COX-2 expression mainly in HNSCC. The tumor cell transformation induced by EGF was repressed with COX-2 knockdown, and this repression was reversed by simultaneously treating the cells with EGF and prostaglandin E2 (PGE2). The down-regulation of COX-2 expression or inhibition of COX-2 activity significantly blocked EGF-enhanced cell migration and invasion, but the addition of PGE2 compensated for this blockage in COX-2-knockdown cells. Interestingly, COX-2 depletion inhibited EGF-induced matrix metalloproteinase-1 (MMP-1), MMP3 and fibronectin expression and Rac1/cdc42 activation; this reduction in MMPs and the fibronectin/Rac1/cdc42 axis by the depletion of COX-2 was also rescued when the cells were treated with PGE2. Furthermore, the depletion of fibronectin impeded the COX-2-enhanced binding of HNSCC cells to endothelial cells and tumor cells metastatic seeding of the lungs. These results provide new insight that EGF-induced COX-2 enhances HNSCC metastasis via activation of the fibronectin signaling pathway. The inhibition of COX-2 expression and activation suggests a potential strategy for the treatment of EGFR-mediated HNSCC metastasis. Citation Format: Jinn-Yuan Hsu, Kwang-Yu Chang, Shang-Hung Chen, Chung-Ta Lee, Sheng-Tsung Chang, Hung-Chi Cheng, Wen-Chang Chang, Ben-Kuen Chen. Epidermal growth factor-induced cyclooxygenase-2 enhances head and neck squamous cell carcinoma metastasis through fibronectin up-regulation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4149. doi:10.1158/1538-7445.AM2015-4149

Nucleolin, expressed in various cellular compartments, participates in DNA transcription, ribosomal biogenesis, regulation of RNA stability. It is evidence that the posttranslational cleavage of nucleolin determine the fate of cells, apoptosis or proliferation. However, the contributions of nucleolin accumulation and cleavage to tumor development are still not clear. Herein we found that nucleolin was dramatically increased in lung cancer and correlated with poor prognosis. The overexpressed nucleolin was cleaved to the smaller fragment (55 kDa), identified in human lung cancer and mouse models of lung cancer. To study the activated mechanism found that EGF-mediated EGFR and KRAS activations could enhance nucleolin expression through increasing Sp1-mediated transcription. Furthermore, to clarify the cleavage residue and cutting enzyme found that nucleolin could be cleaved at 255th Asp by MMP7 (255-710 a.a., TNCL). By using overexpression of TNCL indicated that TNCL enhanced proliferation and metastasis in vitro and in vivo. To further clarify the mechanism of TNCL-enhanced tumor malignancy show that TNCL significantly increase several oncogenes expression including MMP9, Alk and CD74 through associating with 3′-UTR to enhance mRNA stability. Taken together, MMP7 can cleave nucleolin to increase the oncogenes expression, leading lung cancer formation Citation Format: Jan-Jong Hung, Wen-Chang Chang. MMP7-mediated cleavage of nucleolin at the Asp255 induces MMP9 expression to promote tumor malignancy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2089. doi:10.1158/1538-7445.AM2014-2089

The expression of cytokines, such as IL-1β, and the activation of the epidermal growth factor receptor (EGFR) are crucial regulators in the process of carcinogenesis. The correlation between growth factor and activated cytokine signals in the control of tumor development is a critical issue to be clarified. In our study, we found that the IL-1β gene and protein expression were induced by EGF in squamous cell carcinoma. To clarify the mechanism involved in EGF-regulated IL-1β expression, we examined the transcriptional activity and mRNA stability of IL-1β in EGF-treated cells. We found that EGF induced the expression of IL-1β, and was mediated through transcriptional activation, but not through mRNA stability. The involvement of Akt and NF-κB signaling pathways in the EGF-induced IL-1β gene expression and promoter activity was confirmed by treating cells with Akt and NF-κB inhibitors, LY294002 and parthenolide, respectively. Using immunofluorescence staining assay, the EGF-stimulated nuclear translocation of NF-κB (p65) was inhibited by pre-treating cells with LY294002 and parthenolide. Furthermore, EGF increased the binding of NF-κB to the NF-κB binding site of the IL-1β promoter through the activation of the Akt/NF-κB pathway, which resulted in activating IL-1β promoter activity. The expression and secretion of IL-1β induced by EGF considerably reduced chemotherapeutic drug cisplatin-induced cell death. These results showed that EGF enhanced the expression of IL-1β, which was mediated by the Akt/NF-κB pathway. The activation of EGF signaling and increase of IL-1β contributed to chemotherapeutic resistance of cancer cells, suggesting that the expression of IL-1β may be used as a biomarker to evaluate successful cancer treatment. Citation Format: Chi-Ruei Huang, Jhen-Hong Yang, Wen-Chang Chang, Ben-Kuen Chen. Epidermal growth factor protects squamous cell carcinoma against cisplatin-induced cytotoxicity through increased interleukin-1β expression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 999. doi:10.1158/1538-7445.AM2013-999

ZBRK1, a zinc finger protein that interacts with breast cancer 1 (BRCA1) and KRAB-ZFP-associated protein 1 (KAP1), has been suggested to serve as a tumor suppressor via repression of tumor metastasis/invasion. To date, the detailed molecular mechanisms for how BRCA1 and KAP1 participate in ZBRK1-mediated transcriptional repression, metastasis and invasion as well as the associated clinical relevance remain unclear. In this study, we demonstrated that both the N- and C-terminal domains of ZBRK1 are important for inhibiting cell proliferation and anchorage-independent growth in cervical cancer. Specifically, the N-terminal KRAB domain of ZBRK1 displayed a more crucial role in inhibiting metastasis and invasion through modulation of KAP1 function in a transcriptionally dependent manner. The loss of ZBRK1 results in an increase of KAP1 expression, which enhanced migration and invasion of cervical cancer cells both the in vitro and in vivo. Moreover, an inverse correlation of expression levels was observed between ZBRK1 and KAP1 following tumor progression from in situ carcinoma to invasive/metastatic cervical cancer specimens. Taken together, the current results indicate that a loss of ZBRK1 contributes to the increased expression of KAP1, potentiating its role to enhance metastasis and invasion. Citation Format: Wei-Jan Wang, Li-Fang Lin, Chien-Feng Li, Wen-Ming Yang, Dennis Ding-Hwa Wang, Wen-Chang Chang, Wen-Hwa Lee, Ju-Ming Wang, Ju-Ming Wang. Loss of ZBRK1 contributes to the increase of KAP1 and promotes KAP1-mediated metastasis and invasion in cervical cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4926. doi:10.1158/1538-7445.AM2013-4926

The (ARNT) belongs to the basic-helix-loop-helix (bHLH) transcription factors containing Per-Arnt-Sim (PAS) domain. In addition to forming heterodimers with many other bHLH-PAS proteins, including the aryl hydrocarbon receptor (AhR) and hypoxia-inducible factor 1α, ARNT can also form homodimers. Our previous study had shown that ARNT is a new factor involved in EGF regulated COX-2 gene expression under normoxia involving tumorigenesis in cervical cancer tissues. Resistence to cisplatin is often observed in cervical cancer therapy. Here we showed that ARNT expression is critical for cisplatin resistance. Specifically, we showed that in HeLa cervical cancer lines, cisplatin decreased ARNT expression level. Over-expression of ARNT rendered HeLa cells resistant to cisplatin. In addition, inhibition of ARNT by small interfering RNA silencing increased cisplatin induced cell death by activation of caspase 3 and sensitized drug resistant cells to cisplatin. Using ARNT deficient and ARNT fully expressed cell lines showed that induction of apoptosis by cisplatin correlates with ARNT. Importantly down regulation of ARNT decreased drug efflux pump protein MDR1 expression, suggesting that MDR1 gene expression depends on ARNT activity. In conclusion, ARNT mediates the efflux of cisplatin and has a functional role in cervical cancer cell sensitivity to cisplatin. In a xenograft analysis of SCID mice, cisplatin also efficiently inhibited ARNT-deficient tumor formation. These results suggest that targeting ARNT could overcome cisplatin resistance in human cervical cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4228. doi:1538-7445.AM2012-4228