Chia-Chen Hsu

Changes in DNA methylation are associated with the development of drug resistance in cervical cancer cells

Background and proposeChanges in DNA methylation are associated with changes in somatic cell fate without the alteration of coding sequences. In addition to its use as a traceable biomarker, reversible DNA methylation could also serve as a therapeutic target. In particular, if the development of drug resistance is associated with changes in DNA methylation, then demethylation might reverse the resistance phenotype. The reversion of the drug-resistance might then be feasible if the association between abnormal DNA methylation and the development of drug-resistance could be identified.MethodsMethylation differences between the drug-resistance cervical cancer cell, SiHa, and its derived oxaliplatin-resistant S3 cells were detected by methylation specific microarray. The drug-resistance cells were treated with demethylation agent to see if the resistance phenotype were reversed. Targeted methylation of one of the identified locus in normal cell is expected to recapitulate the development of resistance and a two-component reporter system is adopted to monitor the increase of DNA methylation in live cells.ResultsIn this report, we identified methylation changes, both genome-wide and within individual loci, in the oxaliplatin-resistant cervical cancer cell S3 compared with its parental cell line SiHa. Treatment of S3 with a demethylation agent reversed increases in methylation and allowed the expression of methylation-silenced genes. Treatment with the demethylation agent also restored the sensitivity of S3 to cisplatin, taxol, and oxaliplatin to the same level as that of SiHa. Finally, we found that methylation of the target gene Casp8AP2 is sufficient to increase drug resistance in different cells.ConclusionsThese results suggest that global methylation is associated with the development of drug resistance and could serve as a biomarker and therapeutic target for drug resistance in cervical cancer.

Functional characterization of Trip10 in cancer cell growth and survival

BackgroundThe Cdc42-interacting protein-4, Trip10 (also known as CIP4), is a multi-domain adaptor protein involved in diverse cellular processes, which functions in a tissue-specific and cell lineage-specific manner. We previously found that Trip10 is highly expressed in estrogen receptor-expressing (ER+) breast cancer cells. Estrogen receptor depletion reduced Trip10 expression by progressively increasing DNA methylation. We hypothesized that Trip10 functions as a tumor suppressor and may be involved in the malignancy of ER-negative (ER-) breast cancer. To test this hypothesis and evaluate whether Trip10 is epigenetically regulated by DNA methylation in other cancers, we evaluated DNA methylation of Trip10 in liver cancer, brain tumor, ovarian cancer, and breast cancer.MethodsWe applied methylation-specific polymerase chain reaction and bisulfite sequencing to determine the DNA methylation of Trip10 in various cancer cell lines and tumor specimens. We also overexpressed Trip10 to observe its effect on colony formation and in vivo tumorigenesis.ResultsWe found that Trip10 is hypermethylated in brain tumor and breast cancer, but hypomethylated in liver cancer. Overexpressed Trip10 was associated with endogenous Cdc42 and huntingtin in IMR-32 brain tumor cells and CP70 ovarian cancer cells. However, overexpression of Trip10 promoted colony formation in IMR-32 cells and tumorigenesis in mice inoculated with IMR-32 cells, whereas overexpressed Trip10 substantially suppressed colony formation in CP70 cells and tumorigenesis in mice inoculated with CP70 cells.ConclusionsTrip10 regulates cancer cell growth and death in a cancer type-specific manner. Differential DNA methylation of Trip10 can either promote cell survival or cell death in a cell type-dependent manner.

Targeted Casp8AP2 methylation increases drug resistance in mesenchymal stem cells and cancer cells

Casp8AP2 contains a FLASH functional domain and is critical for the formation of death complex and the relay of death signal into the cells. Genetic defects in Casp8AP2 are associated with several diseases. A CpG island within the Casp8AP2 promoter is differentially regulated during somatic stem cell differentiation, and aberrant DNA methylation within the Casp8AP2 promoter has been reported in cancers. We hypothesized that abnormal DNA methylation of Casp8AP2 promoter might contribute to prolonged cellular survival or drug resistance in cancer. The epigenetic state within the Casp8AP2 promoter was then determined in different cancer cell lines and patient samples by methylation-specific PCR. Targeted Casp8AP2 methylation within normal and tumor cells was performed to see whether methylation promoted drug resistance. We found differential Casp8AP2 methylation among the normal and tumoral samples. Global demethylation in a platinum drug-resistant human gastric cancer cell line reversed Casp8AP2 methylation and diminished drug resistance. Targeted methylation of the Casp8AP2 promoter in somatic stem cells and cancer cells increased their resistance to drugs including platinum drugs. These data demonstrate that methylation within the Casp8AP2 promoter correlates with the development of drug resistance and might serve as a biomarker and treatment target for drug resistance in cancer cells.

ENSA expression correlates with attenuated tumor propagation in liver cancer

Endosulfine alpha (ENSA) is an endogenous ligand of sulfonylurea receptor that was reported to be associated with an ATP-dependent potassium channel that controls insulin release and the onset of type 2 diabetes. ENSA also interacts with microtubule-associated serine/threonine-protein kinase-like (MASTL) to regulate the cell cycle. Previously, we identified ENSA as a possible bivalent gene in mesenchymal stem cells (MSCs) and hypothesized its methylation might determine cellular differentiation and transformation. Because there was no link between aberrant ENSA expression and tumorigenesis, we aimed to determine if ENSA is abnormally regulated in liver cancer and plays a role in liver cancer propagation. The epigenetic states of the ENSA promoter were evaluated in different cancer cell lines and patient samples. ENSA was overexpressed in a liver cancer cell line, and its interaction with MASTL and possible tumor suppression capabilities were also determined in cultured cells and mice. Distinct ENSA promoter methylation was observed in liver cancer (n=100 pairs) and breast cancer (n=100 pairs). ENSA was predominantly hypomethylated in liver cancer but was hypermethylated in breast cancer. Overexpressed ENSA interacts with MASTL and suppresses hepatic tumor growth. We also found that ENSA is hypermethylated in CD90-expressing (CD90(+)) cells compared to CD90 non-expressing (CD90(-)) liver cancer cells. These data reveal ENSA methylation changes during hepatic tumor evolution. Overexpressed ENSA suppresses tumor growth in an established hepatic cell line whereas hypermethylated ENSA might help maintain liver cancer initiating cells.

Methylation of the Tumor Suppressor Genes HIC1 and RassF1A Clusters Independently From the Methylation of Polycomb Target Genes in Colon Cancer

BackgroundMethylation changes within tumor suppressor (TS) genes or polycomb group target (PcG) genes alter cell fates. Chromatin associated with PcG targets is bivalent in stem cells, while TS genes are not normally bivalent. PcG target methylation changes have been identified in tumor stem cells, and abnormal methylation is found in TS genes in cancers. If the epigenetic states of genes influence DNA methylation, then methylation of PcG targets and TS genes may evolve differently during cancer development. More importantly, methylation changes may be part of a sequence in tumorigenesis.MethodsChromatin and methylation states of 4 PcG targets and 2 TS genes were determined in colon cancer cells. The methylation states were also detected in 100 pairs of colon cancer samples. Principle component analysis (PCA) was used to reveal whether TS methylation or PcG methylation was the main methylation change associated with colon cancers.ResultsChromatin and methylation states differ in colon cancer cell lines. The methylation states within PcG targets clustered independently from the methylation states in TS genes, a finding we previously reported in liver cancers. PCA in colon cancers revealed the strongest association with methylation changes in 2 TS genes, HIC1 and RassF1A. Loss of HIC1 methylation correlated with decreased tumor migration.ConclusionsPcG and TS methylation states cluster independently from each other. The deduced principle component correlated better with TS methylation than PcG methylation in colon cancer. Abnormal methylation changes may represent a sequential biomarker profile to identify developing colon cancer.

HIC1 and RassF1A Methylation Attenuates Tubulin Expression and Cell Stiffness in Cancer

Cell stiffness is a potential biomarker for monitoring cellular transformation, metastasis, and drug resistance development. Environmental factors relayed into the cell may result in formation of inheritable markers (e.g., DNA methylation), which provide selectable advantages (e.g., tumor development-favoring changes in cell stiffness). We previously demonstrated that targeted methylation of two tumor suppressor genes, hypermethylated in cancer 1 (HIC1) and Ras-association domain family member 1A (RassF1A), transformed mesenchymal stem cells (MSCs). Here, transformation-associated cytoskeleton and cell stiffness changes were evaluated. Atomic force microscopy (AFM) was used to detect cell stiffness, and immunostaining was used to measure cytoskeleton expression and distribution in cultured cells as well as in vivo. HIC1 and RassF1A methylation (me_HR)-transformed MSCs developed into tumors that clonally expanded in vivo. In me_HR-transformed MSCs, cell stiffness was lost, tubulin expression decreased, and F-actin was disorganized; DNA methylation inhibitor treatment suppressed their tumor progression, but did not fully restore their F-actin organization and stiffness. Thus, me_HR-induced cell transformation was accompanied by the loss of cellular stiffness, suggesting that somatic epigenetic changes provide inheritable selection markers during tumor propagation, but inhibition of oncogenic aberrant DNA methylation cannot restore cellular stiffness fully. Therefore, cell stiffness is a candidate biomarker for cells’ physiological status.

Abstract 2409: Loss ofL1TD1suppresses Notch1 inducedLINE1methylation and genomic stability

The silencing of endogenous retrotransposon like LINE1 is critical for the maintenance of genomic stability and the LINE-1 type transposase domain containing 1 (L1TD1) gene possesses the repeated, putative LINE-1 RNA-binding domains and was hypothesized to regulate the activity of LINE-1 through DNA methylation. To verify if L1TD1 is responsible for LINE-1 silencing and the maintenance of somatic genome stability, CRSPR/Cas9 system was used to knockout (KO) L1TD1 locus in gastric, colon and breast cancer cells so as in the mesenchymal stem cells (MSCs). We observed that L1TD1 KO blocked the entrance of SUV39H1, histone methyl transferase, into cell nuclear which suppressed the tri-methylation of histone 3 at lysine 9 (H3k9me3) and LINE-1 methylation. A global demethylation and reduced HP1α recruitment were also observed. The global distribution of CTCF binding loci was also distorted as well as the bivalent histone marks. Further, L1TD1 KO distorted normal distribution of RassF1A expression, cytoskeleton conformation and therefore cell stiffness. The MSC-to-neuron differentiation was also blocked by the KO. Further, we found that overexpressed Notch-1 increased DNA methylation within LINE1 promoter, and this increase was attenuated by L1TD1 KO. Therefore, we conclude that the external signals like Notch1 affect L1NE1 methylation and genome stability through possible LINE1 interacting L1TD1. Since L1TD1 hypermethylation was observed in colon (n=100), gastric (n=19) and breast (n=79) cancers, L1TD1 abnormality is then a candidate for cancer biomarker. (Supported by: MOST-105-2320-B-194-004, MOST-105-2314-B-182A-134, MOST Taiwan and CMRPG6F0092, Chang Gung Memorial Hospital, Chia-Yi, Taiwan) Citation Format: Chia-Chen Chiu, Wei-Chen Huang, Kuan-Der Lee, Chih-Cheng Chen, Chia-Chen Hsu, Mei-Ling Kang, Yu-Wei Leu, Shu-Huei Hsiao. Loss of L1TD1 suppresses Notch1 induced LINE1 methylation and genomic stability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2409. doi:10.1158/1538-7445.AM2017-2409

Abstract 603: Targeted Casp8AP2 methylation increased drug resistance in mesenchymal stem cell and cancer cells

Casp8AP2 is a FLASH protein that is critical for the formation of death complex and the relay of death signal into the cells. Genetic defect of Casp8AP2 was found in diseases like acute lymphoblastic leukemia, implying that the miss-expression of Casp8AP2 might cause disease. On the other hand, the promoter of Casp8AP2 is enriched with CpG islands and we observed differential methylation changes within Casp8AP2 promoter during mesenchymal stem cell (MSCs) differentiation. Aberrant DNA methylation was also identified within the Casp8AP2 promoter in vivo like in liver cancers. It was then hypothesized that the abnormal DNA methylation within the Casp8AP2 promoter might contribute to the enhanced survival or drug resistance in cancer development. We determined the epigenetic states within the Casp8AP2 promoter in different cells and confirmed that there are methylation differences between the normal and tumoral samples within the Casp8AP2 promoter. Differential methylation was also observed between the drug resistant cancer cells and primary cancer cells. Global demethylation in drug resistant cell lines reversed the Casp8AP2 methylation, which was correlated with the reversed resistance phenotype. We then target-methylated the Casp8AP2 promoter in normal somatic stem cells and cancer cells like MDA-MB-231 and found increased drug resistance in these cells. These data point out that methylation within the death complex like Casp8AP2 might be able to serve as biomarker and treatment target for drug resistance in cancer cells. (Supported in part by NSC100-2320-B-194-001) 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 603. doi:1538-7445.AM2012-603

Abstract 4915: Global epigenomic modification platform

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC DNA methylation is an epigenetic mechanism that governs gene silencing and this silencing could be reversed. Pre-deposited global DNA methylation during differentiation is associated with cell fate determination and abnormal DNA methylation could lead to tumorigenesis. A global DNA methylation manipulation system is thus hypothesized to control the differentiation and tumorigenesis more efficiently. Two platforms were established to loss or gain of DNA methylation genome-wide. For the loss of DNA methylation, an in vivo methylation monitoring system was employed to screen 169 procainamide derivatives and identified three distinct classes of these compounds based on their cytotoxicity and demethylation potency. For the gain of DNA methylation, a global DNA methylation subtraction method was developed to identify and methylate target loci genome-wide. After targeted methylation globally, the drug resistance of cancer cells was reverted to become drug sensitive. The same method was used to identify and methylate the hypomethylated loci within somatic stem cells when compared with the differentiated neuronal cells. The targeted methylation accelerated the differentiation of the somatic stem cell to become neuron-like. With both platforms, we wish to evaluate if global DNA methylation changes is sufficient to steer the cell fate. (Supported in part by NSC 97-2320-B-194-003-MY3, NSC 98-3112-B-194-001 and NSC 97-2627-B-006-003) 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 4915.