Eun Ryoung Jang

Development of peptide-based reversing agents for P-glycoprotein-mediated resistance to carfilzomib

Carfilzomib is a novel class of peptidyl epoxyketone proteasome inhibitor and has demonstrated promising activity in multiple clinical trials to treat patients with multiple myeloma and other types of cancers. Here, we investigated molecular mechanisms underlying acquired resistance to carfilzomib and a potential strategy to restore cellular sensitivity to carfilzomib. H23 and DLD-1 cells (human lung and colon adenocarcinoma cell lines) with acquired resistance to carfilzomib displayed marked cross-resistance to YU-101, a closely related proteasome inhibitor, and paclitaxel, a known substrate of Pgp. However, carfilzomib-resistant cells remained sensitive to bortezomib, a clinically used dipeptide with boronic acid pharmacophore. In accordance with these observations, carfilzomib-resistant H23 and DLD-1 cells showed marked upregulation of P-glycoprotein (Pgp) as compared to their parental controls, and coincubation with verapamil, a Pgp inhibitor, led to an almost complete restoration of cellular sensitivity to carfilzomib. These results indicate that Pgp upregulation plays a major role in the development of carfilzomib resistance in these cell lines. In developing a potential strategy to overcome carfilzomib resistance, we as a proof of concept prepared a small library of peptide analogues derived from the peptide backbone of carfilzomib and screened these molecules for their activity to restore carfilzomib sensitivity when cotreated with carfilzomib. We found that compounds as small as dipeptides are sufficient in restoring carfilzomib sensitivity. Taken together, we found that Pgp upregulation plays a major role in the development of resistance to carfilzomib in lung and colon adenocarcinoma cell lines and that small peptide analogues lacking the pharmacophore can be used as agents to reverse acquired carfilzomib resistance. Our findings may provide important information in developing a potential strategy to overcome drug resistance.

ATM modulates transcription in response to histone deacetylase inhibition as part of its DNA damage response

Chromatin structure has a crucial role in a diversity of physiological processes, including development, differentiation and stress responses, via regulation of transcription, DNA replication and DNA damage repair. Histone deacetylase (HDAC) inhibitors regulate chromatin structure and activate the DNA damage checkpoint pathway involving Ataxia-telangiectasia mutated (ATM). Herein, we investigated the impact of histone acetylation/deacetylation modification on the ATM-mediated transcriptional modulation to provide a better understanding of the transcriptional function of ATM. The prototype HDAC inhibitor trichostain A (TSA) reprograms expression of the myeloid cell leukemia-1 (MCL1) and Gadd45α genes via the ATM-mediated signal pathway. Transcription of MCL1 and Gadd45α is enhanced following TSA treatment in ATM+ cells, but not in isogenic ATM- or kinase-dead ATM expressing cells, in the ATM-activated E2F1 or BRCA1-dependent manner, respectively. These findings suggest that ATM and its kinase activity are essential for the TSA-induced regulation of gene expression. In summary, ATM controls the transcriptional upregulation of MCL1 and Gadd45α through the activation of the ATM-mediated signal pathway in response to HDAC inhibition. These findings are important in helping to design combinatory treatment schedules for anticancer radio- or chemo-therapy with HDAC inhibitors.

Methyl CpG–Binding Domain Protein 3 Mediates Cancer-Selective Cytotoxicity by Histone Deacetylase Inhibitors via Differential Transcriptional Reprogramming in Lung Cancer Cells

Histone deacetylase inhibitors (HDI) have been reported to inhibit the growth and survival of cancer cells while leaving normal cells untouched. However, the mechanisms underlying this selective cell death are poorly understood. Gene expression analysis revealed that HDI treatment induced up-regulation of p21(WAF1/Cip1) and down-regulation of ErbB2 in cancer cells but not normal cells. Overexpression of p21(WAF1/Cip1) and/or silencing of ErbB2 enhanced cancer cell growth inhibition, suggesting that HDI-induced up-regulation/down-regulation of these genes play critical roles in HDI-induced growth inhibition of cancer cells. Most importantly, we found that the gene silencing factor methyl CpG-binding domain protein 3 (MBD3) was not only released from cancer-selective promoter of the HDI up-regulated p21(WAF1/Cip1) gene but also recruited to that of the HDI-down-regulated ErbB2 gene. Furthermore, silencing of MBD3 by small interfering RNA abrogated the HDI-induced gene regulation and growth inhibition in lung cancer but not in normal cells. Together, our results support the critical potential of MBD3 in HDI-induced cancer-selective cell death via cancer differential gene expression.

Analysis of ataxia-telangiectasia mutated (ATM)- and Nijmegen breakage syndrome (NBS)-regulated gene expression patterns

Purpose Ataxia-telangiectasia (A-T) is a progressive, degenerative, complex autosomal recessive disease characterized by cerebellar degeneration, immunodeficiency, premature aging, radiosensitivity, and a predisposition to cancer. Mutations in the ataxia-telangiectasia mutated (atm) gene, which phosphorylates downstream effector proteins, are linked to A-T. One of the proteins phosphorylated by the ATM protein is Nijmegen Breakage Syndrome protein (NBS, p95/nibrin), which was recently shown to be encoded by a gene mutated in the Nijmegen breakage syndrome (nbs), an autosomal recessive disease with a phenotype virtually similar to that of A-T. The similarities in the clinical and cellular features of NBS and A-T have led us to hypothesize that the two corresponding gene products may function in similar ways in the cellular signaling pathway. Thus, we sought to identify genes whose expression is mediated by the atm and nbs gene products.Material and methods To identify genes, we performed an analysis of oligonucleotide microarrays using the appropriate cell lines, isogenic A-T (ATM-) and control cells (ATM+), and isogenic NBS (NBS-) and control cells (NBS+).Results We examined genes regulated by ATM and NBS, respectively. To determine the effect of ATM and NBS on gene expression in detail, we classified these genes into different functional categories, including those involved in apoptosis, cell cycle/DNA replication, growth/differentiation, signal transduction, cell-cell adhesion, and metabolism. In addition, we compared the genes regulated by the ATM and NBS to determine the relationship of their signaling pathways and to better understand their functional relationship.Conclusions We found that, while ATM and NBS regulate several genes in common, both of these proteins also have distinct patterns of gene regulation, findings consistent with the functional overlap and distinctiveness of these two conditions. Due to the role of ATM and NBS in tumor suppression and the response to chemotherapy and radiotherapy, these findings may assist in the development of a more rational approach to cancer treatment, as well as a better understanding of tumorigenesis.

A New Isoquinolinium Derivative, Cadein1, Preferentially Induces Apoptosis in p53-defective Cancer Cells with Functional Mismatch Repair via a p38-dependent Pathway

We screened a protoberberine backbone derivative library for compounds with anti-proliferative effects on p53-defective cancer cells. A compound identified from this small molecule library, cadein1 (cancer-selective death inducer 1), an isoquinolinium derivative, effectively leads to a G2/M delay and caspase-dependent apoptosis in various carcinoma cells with non- functional p53. The ability of cadein1 to induce apoptosis in p53-defective colon cancer cells was tightly linked to the presence of a functional DNA mismatch repair (MMR) system, which is an important determinant in chemosensitivity. Cadein1 was very effective in MMR+/p53− cells, whereas it was not effective in p53+ cells regardless of the MMR status. Consistently, when the function of MMR was blocked with short hairpin RNA in SW620 (MMR+/p53−) cells, cadein1 was no longer effective in inducing apoptosis. Besides, the inhibition of p53 increased the pro-apoptotic effect of cadein1 in HEK293 (MMR+/p53+) cells, whereas it did not affect the response to cadein1 in RKO (MMR−/p53+) cells. The apoptotic effects of cadein1 depended on the activation of p38 but not on the activation of Chk2 or other stress-activated kinases in p53-defective cells. Taken together, our results show that cadein1 may have a potential to be an anti-cancer chemotherapeutic agent that is preferentially effective on p53-mutant colon cancer cells with functional MMR.

DNA damage response mediated through BRCA1.

The BRCA1 gene was identified and cloned in 1994 based on its linkage to early onset breast and ovarian cancer syndromes in women. The tumor suppressor, BRCA1 is known as a major player in the DNA damage response. These are evident from its loss, which causes malignant transformation in breast and ovary, and renders cells to become sensitive to a wide variety of DNA damaging agents. Here, we have implications on functional coupling of the pleiotropic roles of BRCA1, including DNA damage signal networking, DNA repair, transcription, and checkpoint of cell cycle, to tumor suppression by examining the molecular mechanisms and functions of BRCA1. n nThe breast cancer susceptibility 1 (BRCA1) gene was identified and mapped to chromosome 17q21 by analyzing families at high risk from breast and ovarian cancer, and was first cloned in 1994 (1). The BRCA1 gene encodes a large nuclear protein that is ubiquitously expressed in a number of tissues. BRCA1 shares little structural resemblance to the majority of other known proteins (Fig. 1). Its ortholog is only found in mammals but not in yeast, fly, worm, or zebra fish, indicating that BRCA1 may come later in evolution and it may have more specialized and tissue-specific functions in mammalian cells. Although a number of studies delineating and deciphering the real biological roles of BRCA1 have accumulated, understanding these BRCA1 unique features still remains to be challengingly elucidated. n n n nFig. 1 n nA schematic diagram of the BRCA1 polypeptide and its interaction with different proteins. BRCA1 polypeptide has the phosphorylation sites (Ⓟ) shown as the seine residues that are phosphorylated and the kinases responsible. The proteins that are ... n n n n nBRCA1 as a tumor suppressor nThe BRCA1 mutations account for about 80% of families whose members have a high incidence of both breast and ovarian cancers (2). The BRCA1 gene fits the profile of a classical tumor suppressor gene, since the breast and ovarian cancers that develop in carriers of BRCA1 gene mutations almost always exhibit loss of the wild-type BRCA1 allele (1). n nThe BRCA1 protein consists of 1863 amino acids (Fig. 1) and is expressed in most proliferating cells (1). The C-terminus of BRCA1 contains an amino-acid sequence motif, now known as a BRCT domain (Fig. 1), recognized in many DNA repair proteins. The BRCT domain of BRCA1 mediates protein interactions that are critical to its role in transcriptional regulation, signaling networking, and the response to DNA damage. The germline mutations in the BRCA1 mutation carriers are frequently found in the BRCT domain, suggesting that the BRCT domain is important for its diverse biological roles. In the N-terminus, there is a ring-finger domain (Fig. 1), also allowing for protein-protein interactions, and thought to be involved in protein ubiquitination. Disruption of the ring domain (by the mutations C61G or C64G) blocked the ability of BRCA1 to repress estrogen receptor-α (ER-α) signaling and to modulate DNA repair, chemosensitivity, and apoptosis. These findings suggest that the ring domain mediates critical protein interactions, and disruption of which contributes to carcinogenesis. Additionally, a variety of structural domains in BRCA1 with these distinct functions hint at diverse roles of BRCA1 in multiple cellular processes. n nRecent epidemiologic studies indicate that BRCA1 mutation carriers have a high lifetime risk of breast cancer of up to 80%. In addition to the breast cancer risk, women with BRCA1 mutations have an increased risk of ovarian cancer. These works solidify the concept that BRCA1 functions as a tumor suppressor. Despite the multiple lines of strong evidences supporting the function of BRCA1 as a tumor suppressor, the mechanisms and the precise roles through which BRCA1 loss leads to tumorigenesis remain to be determined.

Abstract 2611: Inhibition of the immunoproteasome subunit LMP2 by UK-101 leads to apoptosis and cell cycle arrest in pancreatic cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FLnnBackground: The immunoproteasome is an alternative form of the constitutive proteasome and it has been increasingly shown to play an important role in a number of diseases including cancer and inflammatory diseases. We have previously developed ‘UK-101’, a small molecule that selectively inhibits the immunoproteasome catalytic subunit LMP2. In our present study, we investigated the therapeutic potential of the immunoproteasome-targeting approach in pancreatic cancer using UK-101 as a lead compound.nnMethods: The expression of LMP2 was assessed in primary pancreatic cancer tissues and established pancreatic cancer cell lines via immunohistochemical (IHC) and immunoblotting analyses. Using pancreatic cancer cells expressing LMP2, we have investigated the effects of UK-101 (an LMP2 inhibitor), general proteasome inhibitors (bortezomib and carfilzomib) and gemcitabine on cell survival, apoptosis and cell cycle progression. We also examined the effects of UK-101 on various molecular markers associated with apoptosis and cell cycle control.nnResults: Our immunoblotting and IHC analyses indicated that the LMP2 levels are substantially upregulated in primary pancreatic cancer tissues as well as established pancreatic cancer cell lines compared to nonmalignant pancreatic tissues. The IHC staining on a tumor tissue array demonstrated positive immunostaining for LMP2 in approximately 53% of pancreatic cancer specimens evaluated (n=23 out of 43) while the normal pancreatic duct showed a negative LMP2 staining (n=11). Using Panc-1 (gemcitabine-resistant) and BxPc-3 (gemcitabine-sensitive) cells, we observed that the UK-101 treatment leads to a covalent modification of LMP2, a decreased proteasomal proteolytic activity as well as apoptotic cell death and cell cycle arrest.nnConclusion: Our results show that LMP2 is upregulated in pancreatic cancer and that the targeted inhibition of LMP2 using UK-101 induces apoptotic cell death and cell cycle arrest. Taken together, these results suggest that the immunoproteasome may be a potential therapeutic target that can be exploited for pancreatic cancer where the existing therapy including gemcitabine shows limited efficacy.nnCitation 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 2611. doi:10.1158/1538-7445.AM2011-2611

Abstract 2624: Targeted inhibition of the immunoproteasome subunit LMP2 using UK-101 induces apoptotic cell death in colorectal cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnBackground: The immunoproteasome is an alternative form of the constitutive proteasome and it has been increasingly shown to play an important role in a number of diseases including cancer and inflammatory diseases. We have previously developed ‘UK-101’, a small molecule that selectively inhibits the immunoproteasome catalytic subunit LMP2. In our present study, we investigated the frequency of LMP2 expression in primary colon cancers and the impact of targeted inhibition of LMP2 on colon cancer cell survival and apoptosis using UK-101.nnMethods: The levels of LMP2 expression were assessed in colon cancers and adjacent nonmalignant colonic tissues from the matching donors via immunoblotting and immunohistochemical (IHC) analyses. The frequency of LMP2 expression was further determined by performing IHC staining of LMP2 on a tumor tissue microarray (TMA) containing 153 colorectal adenocarcinomas. Using colon cancer cell lines expressing LMP2 (SW480 and DLD-1), we have investigated the effect of UK-101 on tumor cell survival and apoptosis using the MTS and TUNEL assays. To probe the molecular mechanisms of UK-101-induced cell death, we have compared the effects of UK-101 on a number of apoptosis-related proteins and mediators in apoptotic pathways with those of the broadly acting proteasome inhibitors (bortezomib and epoxomicin).nnResults: Our immunoblotting and IHC analyses indicated that the LMP2 levels were substantially upregulated in colon cancers compared to the adjacent nonmalignant colonic tissues from the matching donors. The IHC staining on TMA also demonstrated positive immunostaining for LMP2 in approximately 70% of colon cancer specimens evaluated (n=107 out of 153). A number of colon cancer cell lines were found to express LMP2 at differing levels. Using DLD-1 and SW480 cells expressing LMP2 at moderate to high levels, we observed that UK-101 covalently modifies LMP2, but not other catalytic subunits. The UK-101 treatment also induced cell death, measured by the MTS assay and TUNEL staining. Consistent with apoptotic cell death and proteasomal inhibition, UK-101 treated cells showed the cleavage of PARP and caspase-3 and accumulation of polyubiquitinated proteins. Interestingly, the UK-101 treatment did not show any inhibitory effect on the NF-kB pathway, which was observed with bortezomib and epoxomicin.nnConclusion: Our results show that the targeted inhibition of LMP2 using UK-101 induces apoptotic cell death in colon cancer. UK-101 does not show any inhibitory effect on the NF-kB pathway in colon cancer cells, suggesting that UK-101 induces colon cancer cell apoptosis via mechanisms different from bortezomib and epoxomicin. Taken together, these results suggest that LMP2 may be a potential therapeutic target in treating colon cancer. Further studies are ongoing to investigate the signaling pathways related to UK-101 induced cell death in colon cancer.nnCitation 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 2624.