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PDF file - 287K, Cisplatin-induced enrichment of CD133+ cells

PDF file - 289K, Carboplatin induced DNA damage and CD133+ cell enrichment

PDF file - 342K, Inhibition of cisplatin-induced enrichment of CD133+ cells by Notch1 knockdown

PDF file - 255K, The role of ABC transporters in cisplatin-induced multi-drug resistance to paclitaxol in H460 and H661 cell lines

PDF file - 143K

PDF file - 2.1MB, Cell differentiation and migration of GFP+ cells isolated from H460 cell line

PDF file - 138K, The role of Notch signaling in enrichment of CD133+ cells and multi-drug resistance of H661 cells

Platinum-based chemotherapy is the first-line treatment for non–small cell lung cancer, but recurrence occurs in most patients. Recent evidence suggests that CD133+ cells are the cause of drug resistance and tumor recurrence. However, the correlation between chemotherapy and regulation of CD133+ cells has not been investigated methodically. In this study, we revealed that CD133+ lung cancer cells labeled by a human CD133 promoter–driven GFP reporter exhibited drug resistance and stem cell characteristics. Treatment of H460 and H661 cell lines with low-dose cisplatin (IC20) was sufficient to enrich CD133+ cells, to induce DNA damage responses, and to upregulate ABCG2 and ABCB1 expression, which therefore increased the cross-resistance to doxorubicin and paclitaxel. This cisplatin-induced enrichment of CD133+ cells was mediated through Notch signaling as judged by increased levels of cleaved Notch1 (NICD1). Pretreatment with the γ-secretase inhibitor, N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycine t-butyl ester (DAPT), or Notch1 short hairpin RNAs (shRNA) remarkably reduced the cisplatin-induced enrichment of CD133+ cells and increased the sensitivity to doxorubicin and paclitaxel. Ectopic expression of NICD1 reversed the action of DAPT on drug sensitivity. Immunohistochemistry showed that CD133+ cells were significantly increased in the relapsed tumors in three of six patients with lung cancer who have received cisplatin treatment. A similar effect was observed in animal experiments as cisplatin treatment increased Notch1 cleavage and the ratio of CD133+ cells in engrafted tumors. Intratumoral injection of DAPT with cisplatin treatment significantly reduced CD133+ cell number. Together, our results showed that cisplatin induces the enrichment of CD133+ cells, leading to multidrug resistance by the activation of Notch signaling. Cancer Res; 73(1); 406–16. ©2012 AACR.

Anti-epithelial growth factor receptor (EGFR) monoclonal antibodies and small molecule EGFR inhibitors have been used for treating metastatic colorectal cancer (mCRC) and non-small cell lung cancer (NSCLC), respectively. However, since a relatively large proportion of the mCRC and NSCLC patients carry the activating KRAS/BRAF mutations that can render these EGFR-targeted therapies ineffective, the KRAS/BRAF mutation testing is now required for such prescription. In contrast, epithelial ovarian cancer (EOC) thus far has been treated by surgery, radiotherapy, and/or chemotherapy depending on the tumor stages. However, it has been shown that a high percentage of tumors of the mucinous, endometrioid, low-grade serous, and other types of EOC patients also contain KRAS or BRAF somatic mutations, suggesting that drugs targeting the mediators of the EGFR pathway may have implications on treating EOC of certain types. The conventional analysis of the KRAS, BRAF, and EGFR mutations has been done by multiple reactions with one mutation target per reaction, thus requiring a large amount of precious patient sample for complete testing. Here we present three highly multiplex molecular diagnostic assays, utilizing amplification by allele-specific PCR and automatic detection by a platform designed around the barcoded-magnetic-bead (BMB) suspension-array technology, for detection of 12 KRAS (in codons 12 and 13), six BRAF (in codon 600), and 21 EGFR (in exons 18-21) mutations in mCRC, NSCLC, and EOC patient samples. The results indicate that, albeit all the reaction components are in single wells, these highly multiplex PCR/BMB assays, by comparing with the sequencing results, not only are sensitive and specific but also can conserve precious tissue specimens, save operating time and labor, reduce turnaround time, and increase assay throughput. Citation Format: Jason Lei, Julia Hsu, Peggy Jen, Daniel Huang, Andre Chung, Lloyd Kao, Miller Chang, Chiou-Chung Yuan, Wei-Hwa Lee, Chi-Tai Yeh, Dean Tsao. KRAS, BRAF, and EGFR mutational analysis in ovarian, colon, and lung cancers by highly multiplex PCR/barcoded-magnetic-bead (BMB) suspension-array assays. [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 4663. doi:10.1158/1538-7445.AM2014-4663

Backgrounds: Btk family kinases are non-receptor tyrosine kinases which are involved in the regulation of diverse cellular processes such as growth, survival and migration. Btk up-regulation has been shown to contribute to oncogenesis of myeloid malignacies. However its functions in solid tumors have not been explored due to its prominent expression in B cells. In this study, we present the first evidence that Btk overexpression contributes to breast carcinogenesis. Material and Methods: CTN06-treated and BTK silenced cells were subjected to an Affimetrix human Genome U133A 2.0 microarray analysis for a global examination of Btk's functions in epithelial carcinogenesis. Signaling cascades which were altered by CTN06 treatment include STAT3, mTOR, S6K and Wnt/β-catenin. In vitro proliferation, migration/invasion assays were employed to demonstrate CTN06-mediated anti-cancer effects. RNA interference experiments were performed to show Btk's contribution to breast carcinogenesis and metastasis. Finally, xenograft mouse model was used to demonstrate the effects of CTN06 and the down-regulation of Btk on breast tumorigenesis. Results: Our preliminary data indicates that Btk is aberrantly expressed in various breast cancer cell lines and tissues but particularly in cells of invasive phenotype. These findings prompted us to further explore the role of Btk in breast cancer carcinogenesis. Using available structural models, our team has developed a collection of small molecule inhibitors for Btk and shown that one of them, CTN06, prominently inhibited the growth and migration of different breast cancer cell lines. Interestingly, CTN06 also appeared to be a potent autophagy inducer in the highly invasive and triple-negative MDA-MB-231 cells. Based on these premises and Btk's participation in several signaling cascades such as Src, PI3K, PLCγ and PLC, we hypothesize that Btk overexpression confers a growth and/or survival advantage for breast cancer cells; Btk inhibitors (CTN06) should effectively suppress breast cancer development by negatively modulating Btk-related networks and be evaluated for clinical use. We have obtained promising results not only in vitro cell line models but also in vivo animal xenograft experiments to demonstrate its efficacy and safety profile. Conclusions: We demonstrated for the first time that Btk overexpression is detected in breast cancer cells particularly in metastatic MDA-MB-231 cells and Btk-specific inhibitor CTN06 effectively suppressed both tumor growth and metastasis via negatively modulating the aforementioned signaling cascades in vitro and in vivo. Citation Format: Chih-Ming Su, Chin-Nien Chuang, Alexander Wu, Liang-Shun Wang, Wei-Hwa Lee, Chih-Hsiung Wu, Kit Lam, Hsing-Jien Kung, Chi-Tai Yeh. A novel Btk tyrosine kinase inhibitor, CTN06, as a potential candidate agent for breast cancer treatment. [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 2087. doi:10.1158/1538-7445.AM2013-2087

Cancer stem cells (CSCs) have been defined as a subpopulation of cancer cells with the ability of self-renewal, giving rise to different progenies, and transition between epithelial and mesenchymal status in response to the tumor microenvironment. Due to the lack of an appropriate cell model, our understanding of CSC biology remains poor and the development of CSC antagonists is challenging. We herein used the UP-LN1 cell line as a CSC model which was derived from a gastrointestinal tumor-to-lymph node metastasis lesion. UP-LN1 cells constitute two major components, the floating (F) and adherent (A) cells. F cells were identified as the primary niche of CSCs, and could be serially subcultured while maintaining their stem-like properties including high self-renewal potential, formation of suspended grape-like aggregates and/or spheres, resistance to multiple drugs and NK/LAK effectors, as well as an increased CDY1 (a novel iPS probe) dye retention. Using this cell model, we demonstrated that Withaferin A (WA) dramatically reduced the proliferation of F cells and their ability to form aggregates in vitro. WA also dose-dependently reduced the side population cells. Mechanistically, WA treatment resulted in the down-regulation of two axes, CXCR4 and STAT3, both instrumental in the acquisition of metastatic ability and the development/maintenance of CSCs. We validated the in vitro results using non-invasive imaging technique in a xenograft mouse model. WA-treated animals demonstrated a marked decrease in tumor burden and metastasis, indicating that WA is potentially an effective agent for targeting CSCs. Citation Format: Chi-Tai Yeh, Alexander TH Wu, Hung-Chang Chen, Cheng-Keng Chuang, See-Tong Pang, Shuen-Kuei Liao. Anticancer effects of Withaferin A on UP-LN1 carcinoma cells through the inhibition of STAT3 phosphorylation and IFN-γ-mediated induction of metastatic cancer stem cells . [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 222. doi:10.1158/1538-7445.AM2013-222

Tumor microenvironment plays a key role in promoting epithelial-to-mesenchymal transition (EMT), a cellular step for cancer cells to gain metastatic ability and is also associated to the generation of cancer stem cells (CSCs). Agents capable of modulating the tumor microenvironment could be more efficient in managing tumor progression. Pterostilbene, a natural stilbene isolated from blueberries have been suggested for its anti-cancer effects. Here, we explored the potential microenvironment modulating effects of pterostilbene in a M2-polarized macrophages (M2 TAMs) and breast cancer cell co-culture system. We first demonstrated that co-culturing with M2 TAMs increased the percentage of CD44+/CD24- CSC population and migratory/invasive abilities. We then showed that pterostilbene treatment dose-dependently overcame M2 TAM-induced enrichment of CSCs and metastatic potential of breast cancer cells. Mechanistically, pterostilbene suppressed NFκB, Twist1, Vimentin and increased E-cadherin expression. Importantly, pterostilbene-mediated NFκB downregulation was correlated to an increased amount of microRNA448. Finally, using non-invasive bioluminescence technique we demonstrated that pterostilbene treatment significantly suppressed M2 TAM co-cultured MDA-MB-231 tumorigenesis and metastasis. Thus, pterostilbene could be a potential anti-CSC agent for clinical development. Citation Format: Chi-Tai Yeh, Jeng-Fong Chiou, Chih-Hsiung Wu, Alexander TH Wu. Pterostilbene suppresses the generation of breast cancer stem cells within tumor microenvironment and metastasis via modulating NF-κB/microRNA448 circuit. [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 229. doi:10.1158/1538-7445.AM2013-229

The anticancer effects of honokiol, an active component isolated from Chinese traditional herb Magnolia officinalis, had been noticed in recent decade. It had been reported to induce apoptosis, anti-agiogenesis and chemo/radio-sensitization on cancer cells. However, its effects on autophagy induction and cancer stem cell elimination have not yet been investigated. To more evaluate the potential of honokiol in cancer therapy, we explored its effects on these two critical events in glioblastoma multiforme (GBM) DBTRG-05MG cells. The honokiol-induced autophagy was evidenced by the time and dose-dependent increase of LC3-II in treated cells. During this autophagy induction, the phosphorylated AMP-dependent kinase alpha was dose-dependently increased in accompany with the decrease of phosphorylated mTOR protein level. In addition, the phosphorylated p38 was also markedly increased parallel with the elevation of LC3-II. Further investigation on the role of this MAPK p38 pathway in honokiol-induced autophagy is ongoing. In accordance with the growth inhibition and massive cell death induced by high dose of honokiol (50 μM), the phsphorylated Akt and Rb were dramatically suppressed in treated cells. As it is suggested that autophagy is a protective response of cancer cell to environmental stress, an autophagy inhibitor hydroxychloroquine was therefore co-administrated to enhance the effects of honokiol against GBM cells. Significantly, hydroxychloroquine augmented the honokiol-induced cell death, indicating the crucial role of autophagy in honokiol-induced anticancer effects. Moreover, honokiol appeared to have effects on the elimination of cancer stem-like cells. By UV laser-equipped flow cytometer, a small percentage of cancer stem-like side population (SP) cells was detected and sorted from another GBM cell line (GBM 8401). The expressions of stemness genes such as CD133, nestin, Oct4, NOTCH3, IHH and SMO in SP cells were much higher than those in non-SP cells. Treatment with honokiol (5 μM) for 48h decreased the percentage of GBM 8401 SP cells from 1.5% down to 0.2%. These results suggested the potential role of honokiol in GBM treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3778. doi:10.1158/1538-7445.AM2011-3778

Leukemia is a serious disease that affects both children and adults. Although all-trans retinoic acid (ATRA) and targeted drug such as imatinib could effectively treat acute promyelocytic (APL) and chronic myelogenous leukemia (CML), respectively, rare therapeutics is available when drug resistance or recurrence occurred. Searching for alternative agent for refractory patient is urgently needed. Our previous studies had found that a human urine extract CDA-2 could induce differentiation of HL60 APL cells as well as potentiate the effects of ATRA and imatinib on HL60 and K562 CML cells, respectively. Purification of active compound possessing these anti-leukemic effects from CDA-2 was thus performed. Recently, an active compound named as P18.9 was isolated. It induced differentiation of K562 CML cells as evidenced by the nitroblue-tetrazolium (NBT) reduction test. At higher dose, flow cytometry analysis showed that P18.9 induced sub-G1 apoptotic fraction of K562 cells and enhanced the sub-G1 fraction induced by imatinib at a scale of about two folds. Although P18.9 did not induce differentiation of HL60 APL cells, it substantially enhanced the ATRA-induced differentiation. This is the first study to isolate an active compound from human urine, which exerts anti-leukemic effects alone or cooperatively with clinical used drugs. The chemical structure of P18.9 had been identified and further subsequent structure modification is ongoing. Continuing the effort to develop P18.9 and its derivatives as novel therapeutics for refractory APL or CML patients is warranted. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C189.