Browsing by Author "Lee, Chia-Hwa"

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A rapid and quantitative method to detect human circulating tumor cells in a preclinical animal model
(2017-06-23)
Tu, Shih-Hsin
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Hsieh, Yi-Chen
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Huang, Li-Chi
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Lin, Chun-Yu
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Hsu, Kai-Wen
Background: As cancer metastasis is the deadliest aspect of cancer, causing 90% of human deaths, evaluating the molecular mechanisms underlying this process is the major interest to those in the drug development field. Both therapeutic target identification and proof-of-concept experimentation in anti-cancer drug development require appropriate animal models, such as xenograft tumor transplantation in transgenic and knockout mice. In the progression of cancer metastasis, circulating tumor cells (CTCs) are the most critical factor in determining the prognosis of cancer patients. Several studies have demonstrated that measuring CTC-specific markers in a clinical setting (e.g., flow cytometry) can provide a current status of cancer development in patients. However, this useful technique has rarely been applied in the real-time monitoring of CTCs in preclinical animal models. Methods: In this study, we designed a rapid and reliable detection method by combining a bioluminescent in vivo imaging system (IVIS) and quantitative polymerase chain reaction (QPCR)-based analysis to measure CTCs in animal blood. Using the IVIS Spectrum CT System with 3D-imaging on orthotropic-developed breast-tumor-bearing mice. Results: In this manuscript, we established a quick and reliable method for measuring CTCs in a preclinical animal mode. The key to this technique is the use of specific human and mouse GUS primers on DNA/RNA of mouse peripheral blood under an absolute qPCR system. First, the high sensitivity of cancer cell detection on IVIS was presented by measuring the luciferase carried MDA-MB-231 cells from 5 to 5x10(11) cell numbers with great correlation (R-2 = 0.999). Next, the MDA-MB-231 cell numbers injected by tail vein and their IVIS radiance signals were strongly corrected with qPCR-calculated copy numbers (R-2 > 0.99). Furthermore, by applying an orthotropic implantation animal model, we successfully distinguished xenograft tumor-bearing mice and control mice with a significant difference (p < 0.001), whereas IVIS Spectrum-CT 3D-visualization showed that blood of mice with lung metastasis contained more than twice the CTC numbers than ordinary tumor-bearing mice. We demonstrated a positive correlation between lung metastasis status and CTC numbers in peripheral mouse blood. Conclusion: Collectively, the techniques developed for this study resulted in the integration of CTC assessments into preclinical models both in vivo and ex vivo, which will facilitate translational targeted therapy in clinical practice.
ABLGenomic Editing Sufficiently Abolishes Oncogenesis of Human Chronic Myeloid Leukemia Cells In Vitro and In Vivo
(2020-06-01)
Chen, Shu-Huey
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Hsieh, Yao-Yu
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Tzeng, Huey-En
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Lin, Chun-Yu
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Hsu, Kai-Wen
Chronic myelogenous leukemia (CML) is the most common type of leukemia in adults, and more than 90% of CML patients harbor the abnormal Philadelphia chromosome (Ph) that encodes the BCR-ABL oncoprotein. Although the ABL kinase inhibitor (imatinib) has proven to be very effective in achieving high remission rates and improving prognosis, up to 33% of CML patients still cannot achieve an optimal response. Here, we used CRISPR/Cas9 to specifically target theBCR-ABLjunction region in K562 cells, resulting in the inhibition of cancer cell growth and oncogenesis. Due to the variety ofBCR-ABLjunctions in CML patients, we utilized gene editing of the humanABLgene for clinical applications. Using theABLgene-edited virus in K562 cells, we detected 41.2% indels inABLsgRNA_2-infected cells. TheABL-edited cells reveled significant suppression of BCR-ABL protein expression and downstream signals, inhibiting cell growth and increasing cell apoptosis. Next, we introduced theABLgene-edited virus into a systemic K562 leukemia xenograft mouse model, and bioluminescence imaging of the mice showed a significant reduction in the leukemia cell population inABL-targeted mice, compared to the scramble sgRNA virus-injected mice. In CML cells from clinical samples, infection with theABLgene-edited virus resulted in more than 30.9% indels and significant cancer cell death. Notably, no off-target effects or bone marrow cell suppression was found using theABLgene-edited virus, ensuring both user safety and treatment efficacy. This study demonstrated the critical role of theABLgene in maintaining CML cell survival and tumorigenicity in vitro and in vivo.ABLgene editing-based therapy might provide a potential strategy for imatinib-insensitive or resistant CML patients.
An Integrated Genomic Strategy to Identify CHRNB4 as a Diagnostic/Prognostic Biomarker for Targeted Therapy in Head and Neck Cancer
(2020-05-01)
Chuang, Yi-Hsuan
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Lee, Chia-Hwa
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Lin, Chun-Yu
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Liu, Chia-Lin
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Huang, Sing-Han
Although many studies have shown the association between smoking and the increased incidence and adverse prognosis of head and neck squamous cell carcinoma (HNSCC), the mechanisms and pharmaceutical targets involved remain unclear. Here, we integrated gene expression signatures, genetic alterations, and survival analyses to identify prognostic indicators and therapeutic targets for smoking HNSCC patients, and we discovered that the FDA-approved drug varenicline inhibits the target for cancer cell migration/invasion. We first identified 18 smoking-related and prognostic genes for HNSCC by using RNA-Seq and clinical follow-up data. One of these genes, CHRNB4 (neuronal acetylcholine receptor subunit beta-4), increased the risk of death by approximately threefold in CHRNB4-high expression smokers compared to CHRNB4-low expression smokers (log rank, p = 0.00042; hazard ratio, 2.82; 95% CI, 1.55-5.14), former smokers, and non-smokers. Furthermore, we examined the functional enrichment of co-regulated genes of CHRNB4 and its 246 frequently occurring copy number alterations (CNAs). We found that these genes were involved in promoting angiogenesis, resisting cell death, and sustaining proliferation, and contributed to much worse outcomes for CHRNB4-high patients. Finally, we performed CHRNB4 gene editing and drug inhibition assays, and the results validate these observations. In summary, our study suggests that CHRNB4 is a prognostic indicator for smoking HNSCC patients and provides a potential new therapeutic drug to prevent recurrence or distant metastasis.
CRISPR/Cas9 Genome Editing of Epidermal Growth Factor Receptor Sufficiently Abolished Oncogenicity in Anaplastic Thyroid Cancer
(2018-01-01)
Huang, Li-Chi
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Tam, Ka-Wai
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Liu, Wei-Ni
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Lin, Chun-Yu
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Hsu, Kai-Wen
Anaplastic carcinoma of the thyroid (ATC), also called undifferentiated thyroid cancer, is the least common but most aggressive and deadly thyroid gland malignancy of all thyroid cancers. The aim of this study is to explore essential biomarker and use CRISPR/Cas9 with lentivirus delivery to establish a gene-target therapeutic platform in ATC cells. At the beginning, the gene expression datasets from 1036 cancers from CCLE and 8215 tumors from TCGA were collected and analyzed, showing EGFR is predominantly overexpressed in thyroid cancers than other type of cancers (P = 0 017 in CCLE and P = 0 001 in TCGA). Using CRISPR/Cas9 genomic edit system, ATC cells with EGFR sgRNA lentivirus transfection obtained great disruptions on gene and protein expression, resulting in cell cycle arrest, cell growth inhibition, and most importantly metastasis turn-off ability. In addition, the FDA-approved TKI of afatinib for EGFR targeting also illustrates great anticancer activity on cancer cell death occurrence, cell growth inhibition, and cell cycle arrest in SW579 cells, an EGFR expressing human ATC cell line. Furthermore, off-target effect of using EGFR sgRNAs was measured and found no genomic editing can be detected in off-target candidate gene. To conclude, this study provides potential ATC therapeutic strategies for current and future clinical needs, which may be possible in increasing the survival rate of ATC patients by translational medicine.
HDAC1 and HDAC2 Double Knockout Triggers Cell Apoptosis in Advanced Thyroid Cancer
(2019-01-02)
Lin, Ching-Ling
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Tsai, Ming-Lin
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Lin, Chun-Yu
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Hsu, Kai-Wen
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Hsieh, Wen-Shyang
Anaplastic thyroid carcinoma (ATC) and squamous thyroid carcinoma (STC) are both rare and advanced thyroid malignancies with a very poor prognosis and an average median survival time of 5 months and less than 20% of affected patients are alive 1 year after diagnosis. The clinical management of both ATC and STC is very similar because they are not particularly responsive to radiotherapy and chemotherapy. This inspired us to explore a novel and effective clinically approved therapy for ATC treatment. Histone deacetylase inhibitor (HDACi) drugs are recently FDA-approved drug for malignancies, especially for blood cell cancers. Therefore, we investigated whether an HDACi drug acts as an effective anticancer drug for advanced thyroid cancers. Cell viability analysis of panobinostat treatment demonstrated a significant IC50 of 0.075 mu M on SW579 STC cells. In addition, panobinostat exposure activated histone acetylation and triggered cell death mainly through cell cycle arrest and apoptosis-related protein activation. Using CRISPR/Cas9 to knock out HDAC1 and HDAC2 genes in SW579 cells, we observed that the histone acetylation level and cell cycle arrest were enhanced without any impact on cell growth. Furthermore, HDAC1 and HDAC2 double knockout (KO) cells showed dramatic cell apoptosis activation compared to HDAC1 and HDAC2 individual KO cells. This suggests expressional and biofunctional compensation between HDAC1 and HDAC2 on SW579 cells. This study provides strong evidence that panobinostat can potentially be used in the clinic of advanced thyroid cancer patients.
HDAC1,2 Knock-Out and HDACi Induced Cell Apoptosis in Imatinib-Resistant K562 Cells
(2019-05-01)
Chen, Shu-Huey
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Chow, Jyh-Ming
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Hsieh, Yao-Yu
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Lin, Chun-Yu
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Hsu, Kai-Wen
Since imatinib (Glivec or Gleevec) has been used to target the BCR-ABL fusion protein, chronic myeloid leukemia (CML) has become a manageable chronic disease with long-term survival. However, 15%-20% of CML patients ultimately develop resistance to imatinib and then progress to an accelerated phase and eventually to a blast crisis, limiting treatment options and resulting in a poor survival rate. Thus, we investigated whether histone deacetylase inhibitors (HDACis) could be used as a potential anticancer therapy for imatinib-resistant CML (IR-CML) patients. By applying a noninvasive apoptosis detection sensor (NIADS), we found that panobinostat significantly enhanced cell apoptosis in K562 cells. A further investigation showed that panobinostat induced apoptosis in both K562 and imatinib-resistant K562 (IR-K562) cells mainly via H3 and H4 histone acetylation, whereas panobinostat targeted cancer stem cells (CSCs) in IR-K562 cells. Using CRISPR/Cas9 genomic editing, we found that HDAC1 and HDAC2 knockout cells significantly induced cell apoptosis, indicating that the regulation of HDAC1 and HDAC2 is extremely important in maintaining K562 cell survival. All information in this study indicates that regulating HDAC activity provides therapeutic benefits against CML and IR-CML in the clinic.
Membrane protein-regulated networks across human cancers
(2019-07-16)
Lin, Chun-Yu
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Lee, Chia-Hwa
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Chuang, Yi-Hsuan
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Lee, Jung-Yu
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Chiu, Yi-Yuan
Alterations in membrane proteins (MPs) and their regulated pathways have been established as cancer hallmarks and extensively targeted in clinical applications. However, the analysis of MP-interacting proteins and downstream pathways across human malignancies remains challenging. Here, we present a systematically integrated method to generate a resource of cancer membrane protein-regulated networks (CaMPNets), containing 63,746 high-confidence protein-protein interactions (PPIs) for 1962 MPs, using expression profiles from 5922 tumors with overall survival outcomes across 15 human cancers. Comprehensive analysis of CaMPNets links MP partner communities and regulated pathways to provide MP-based gene sets for identifying prognostic biomarkers and druggable targets. For example, we identify CHRNA9 with 12 PPIs (e.g., ERBB2) can be a therapeutic target and find its anti-metastasis agent, bupropion, for treatment in nicotine-induced breast cancer. This resource is a study to systematically integrate MP interactions, genomics, and clinical outcomes for helping illuminate cancer-wide atlas and prognostic landscapes in tumor homo/heterogeneity.
Nicotinic Acetylcholine Receptor Subtype Alpha-9 Mediates Triple-Negative Breast Cancers Based on a Spontaneous Pulmonary Metastasis Mouse Model
(2017-11-03)
Huang, Li-Chi
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Lin, Ching-Ling
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Qiu, Jia-Zheng
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Lin, Chun-Yu
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Hsu, Kai-Wen
Triple-negative breast cancer (TNBC) subtype is associated with poor prognosis and a high risk of recurrence-related death in women. Despite the aggressiveness of TNBCs, targeted TNBC therapy is not yet available in the clinic. To overcome this challenge, we generated highly metastatic TNBC cells (LM) derived from metastasized lung cells via a serial spontaneous pulmonary metastasis animal model to identify targetable molecules for attenuating the progression of TNBC metastasis. Gene analysis of primary tumor (P), first-round (1LM) and second-round (2LM) metastasized lung cells revealed that mesenchymal-related genes were significantly expressed in LM cells, especially in 2LM cells. Interestingly, alpha 9-nAChR gene expression was also dramatically induced in LM cells, confirming our previous finding that alpha 9-nAChR plays important roles in receptor-mediated carcinogenic signals in human breast cancer development. Using alpha 9-nAChR as a biomarker, we transfected 2LM cells with CRISPR/Cas9 lentivirus targeting the alpha 9-nAChR genomic region (2LM-alpha 9-nAChR-null), showing that mesenchymal markers and the migration and invasion abilities of 2LM cells were significantly attenuated in 2LM-alpha 9-nAChR-null cells both in vitro and in vivo. In addition, the high efficiency of editing the alpha 9-nAChR gene using a CRISPR/Cas9 lentivirus was demonstrated by gene sequencing, genomic indel frequency and protein expression analyses. Collectively, these results confirmed those of our previous study that advanced-stage breast tumors are associated with substantially higher levels of alpha 9-nAChR gene expression, indicating that alpha 9-nAChR expression is essential for mediating TNBC metastasis during cancer development and may potentially act as a biomarker for targeted therapy in clinical investigations.
The Application of Non-Invasive Apoptosis Detection Sensor (NIADS) on Histone Deacetylation Inhibitor (HDACi)-Induced Breast Cancer Cell Death
(2018-02-01)
Hsu, Kai-Wen
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Huang, Chien-Yu
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Tam, Ka-Wai
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Lin, Chun-Yu
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Huang, Li-Chi
Breast cancer is the most common malignancy in women and the second leading cause of cancer death in women. Triple negative breast cancer (TNBC) subtype is a breast cancer subset without ER (estrogen receptor), PR (progesterone receptor) and HER2 (human epidermal growth factor receptor 2) expression, limiting treatment options and presenting a poorer survival rate. Thus, we investigated whether histone deacetylation inhibitor (HDACi) could be used as potential anti-cancer therapy on breast cancer cells. In this study, we found TNBC and HER2-enriched breast cancers are extremely sensitive to Panobinostat, Belinostat of HDACi via experiments of cell viability assay, apoptotic marker identification and flow cytometry measurement. On the other hand, we developed a bioluminescence-based live cell non-invasive apoptosis detection sensor (NIADS) detection system to evaluate the quantitative and kinetic analyses of apoptotic cell death by HDAC treatment on breast cancer cells. In addition, the use of HDACi may also contribute a synergic anti-cancer effect with co-treatment of chemotherapeutic agent such as doxorubicin on TNBC cells (MDA-MB-231), but not in breast normal epithelia cells (MCF-10A), providing therapeutic benefits against breast tumor in the clinic.