Daeil Sung

Disulfiram targets cancer stem-like properties and the HER2/Akt signaling pathway in HER2-positive breast cancer

HER2-positive breast tumors are known to harbor cancer stem-like cell populations and are associated with an aggressive tumor phenotype and poor clinical outcomes. Disulfiram (DSF), an anti-alcoholism drug, is known to elicit cytotoxicity in many cancer cell types in the presence of copper (Cu). The objective of the present study was to investigate the mechanism of action responsible for the induction of apoptosis by DSF/Cu and its effect on cancer stem cell properties in HER2-positive breast cancers in vitro and in vivo. DSF/Cu treatment induced apoptosis, associated with a marked decrease in HER2, truncated p95HER2, phospho-HER2, HER3, phospho-HER3 and phospho-Akt levels, and p27 nuclear accumulation. This was accompanied by the eradication of cancer stem-like populations, concomitant with the suppression of aldehyde dehydrogenase 1 (ALDH1) activity and mammosphere formation. DSF administration resulted in a significant reduction in tumor growth and an enhancement of apoptosis, as well as HER2 intracellular domain (ICD) and ALDH1A1 downregulation. Our results demonstrate that DSF/Cu induces apoptosis and eliminates cancer stem-like cells via the suppression of HER2/Akt signaling, suggesting that DSF may be potentially effective for the treatment of HER2-positive cancers.

Disulfiram induces anoikis and suppresses lung colonization in triple-negative breast cancer via calpain activation

Triple-negative breast cancers (TNBC) often exhibit an aggressive phenotype. Disulfiram (DSF) is an approved drug for the treatment of alcohol dependence, but has also been shown to kill TNBC cells in a copper (Cu)-dependent manner. Exactly how this occurs has not been clearly elucidated. We sought to investigate the mechanisms responsible for DSF/Cu-dependent induction of apoptosis and suppression of lung colonization by TNBC cells. DSF/Cu induced anoikis and significantly suppressed cell migration and invasion with negative effects on focal adhesions, coinciding with vimentin breakdown and calpain activation in TNBC cells. In a xenograft tumor model, DSF suppressed tumor growth and lung nodule growth, which was also associated with calpain activation. These findings warrant further investigation of disulfiram as a potential treatment for metastatic TNBC.

Inhibition of ubiquitin-specific protease 34 (USP34) induces epithelial-mesenchymal transition and promotes stemness in mammary epithelial cells

Ubiquitin-specific protease 34 (USP34) is a deubiquitinating enzyme that regulates Axin stability and plays a critical role in Wnt/β-catenin signaling. We sought to investigate the role of USP34 on epithelial-mesenchymal (EMT) induction and its effects on mammary epithelial stem cells. USP34 expression levels were relatively lower in MDA-MB-231 and 4T1 mesenchymal-like cells when compared to epithelial-like cells. Inhibition of USP34 in NMuMG cells induced EMT, as evidenced by the upregulation of EMT markers including N-cadherin, phospho-Smad3, Snail and active-β-catenin, as well as the downregulation of Axin 1 and E-cadherin. USP34 knockdown (KD) in these cells also resulted in the acquisition of invasive behavior, and promoted stemness as indicated by enhanced mammosphere-forming ability, concomitant with the upregulation of Nanog, Oct4 and Sox2 mRNA expression. Endogenous USP34 expression was observed to be at low levels in virgin mouse mammary glands in vivo. When USP34-KD cells were transplanted into the cleared mammary fat pads (CFP) of mice, these cells reconstituted the mammary gland with ductal tree development within 3months. Our findings suggest a previously unknown role for USP34 in mammary gland development.

Flubendazole elicits anti-metastatic effects in triple-negative breast cancer via STAT3 inhibition: Flubendazole suppresses metastasis via STAT3 inhibition

Tumor metastasis remains the cause of 90% of cancer‐related deaths. Cancer stem cells (CSC) are thought to be responsible for the aggressive and metastatic nature of triple‐negative breast cancers (TNBC), and new therapeutic strategies are being devised to target them. Flubendazole (FLU) is a widely used anthelmintic agent that also exhibits anticancer activity in several cancer types. The aim of this study was to characterize the mechanism of action of FLU on breast cancer stem cell (BCSC)‐like properties and metastasis in TNBC. FLU treatment caused a significant induction of apoptosis, accompanied by G2/M phase accumulation, caspase‐3/‐7 activation and the dysregulation of STAT3 activation in TNBC cells. The latter phenomenon was associated with impairment of cancer stem‐like traits, concomitant with a reduction in the CD24low/CD44high, CD24high/CD49fhigh subpopulation, ALDH1 activity and mammosphere formation. The BCSC‐enriched populations exhibited enhanced metastasis with higher STAT3 activation, while FLU administration inhibited tumor growth, angiogenesis and lung and liver metastasis, coinciding with decreased MMP‐2 and MMP‐9 levels in circulating blood. FLU kills not only rapid proliferating tumor cells but also effectively eradicates BCSC‐like cells in vitro and in vivo. Our findings warrant further investigation of FLU as a treatment for metastatic TNBC.

Flubendazole overcomes trastuzumab resistance by targeting cancer stem-like properties and HER2 signaling in HER2-positive breast cancer

Although trastuzumab provides significant clinical benefit for HER2-positive breast cancers, responses are limited by the emergence of resistance. Trastuzumab resistance is a multi-factorial phenomenon thought to arise from the presence of cancer stem cells and interactions between truncated p95HER2 and HER family members. Flubendazole (FLU) is a potent anthelmintic agent with an exceptional safety profile. Evidence also suggests that it can act as an anticancer agent in several cancer cell types. We sought to investigate the effect of FLU on apoptosis, HER2/Akt signaling, breast cancer stem cell (BCSC)-like properties and trastuzumab resistance in HER2-positive breast cancer cells. FLU treatment induced apoptosis, associated with a significant downregulation of truncated p95HER2, phospho-HER2, phospho-HER3 and phospho-Akt levels, as well as suppression of HER2/HER3 hetero-dimerization in both trastuzumab-sensitive and -resistant lines. FLU effectively targeted BCSC-like properties including aldehyde dehydrogenase 1 (ALDH1) expression and the CD44high/CD24low phenotype, concomitant with a suppression of mammosphere-forming ability. FLU administration also caused significant tumor suppression in trastuzumab-resistant xenografts, coinciding with the downregulation of BCSC-related markers and intracellular HER2. These findings highlight the mechanisms of action of FLU in overcoming trastuzumab resistance in breast cancer.

Abstract 5463: Disulfiram suppresses metastasis via induction of anoikis and calpain activation in triple-negative breast cancer

Triple-negative breast cancers (TNBC) exhibit aggressive phenotypes and are associated with poor clinical outcomes due to a lack of effective therapeutic strategies. Disulfiram (DSF) is a clinical therapeutic drug for the treatment of alcoholism, and has also been shown to exhibit antitumor activity in TNBC cells in a copper (Cu)-dependent manner, although how this occurs has not been clearly elucidated. We sought to investigate the mechanisms responsible for DSF/Cu-dependent induction of apoptosis and suppression of lung colonization by TNBC cells. DSF/Cu treatment significantly induced apoptosis, accompanied by an increase in the sub-G1 population, as well as caspase-3 activation. DSF/Cu strongly suppressed cell migration and invasion, while inducing suppression of focal adhesion kinase (FAK) phosphorylation and focal adhesions, concomitant with an observed reorganization of cytoskeletal F-actin and collapse of the plasma membrane. These effects were associated with calpain activation, coinciding with the breakdown of vimentin, FAK and talin, presumably allowing for cell detachment. In a xenograft tumor model, DSF suppressed tumor growth and lung colonization, which was also associated with calpain activation. Taken together, these findings warrant further investigation of disulfiram as a potential treatment for metastatic TNBC. Citation Format: Eunhye Oh, Daeil Sung, Youngkwan Cho, Ji Young Kim, Nahyun Lee, Yoon-Jae Kim, Tae-Min Cho, Jae Hong Seo. Disulfiram suppresses metastasis via induction of anoikis and calpain activation in triple-negative breast cancer [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 5463. doi:10.1158/1538-7445.AM2017-5463

Abstract 4302: Disulfiram possesses antitumor activity and inhibits cancer stem-like properties via inhibition of STAT3 activation in triple-negative breast cancer cells

Disulfiram (DSF), a clinically used treatment for alcohol dependence, is considered as a potential anti-cancer agent due to its cytotoxicity in many cancer cell types on copper (Cu)-dependent processes. However, the molecular mechanisms underlying DSF-induced antitumor activity in triple-negative breast cancer (TNBC) are still poorly understood. Here, we hypothesized that DSF induces antitumor effects by targeting cancer stem cells (CSCs) and STAT3 signaling in TNBC. We then examined the effects of DSF in combination with Cu on the cell viability and apoptosis, cell proliferation and survival, as well as CSC-like properties in MDA-MB-231 and 4T1 TNBC cell lines. DSF/Cu effectively induced cytotoxicity and apoptotic cell death, concomitant with caspase-3 activation and PARP cleavage. DSF/Cu also significantly suppressed cell migration and mammosphere formation, which are accompanied by a remarkable decrease in the CD44+/CD24- (or CD49f+/CD24+ in mouse 4T1 cells) stem-like population and ALDH1 activity. These effects were functionally associated with the inhibition of STAT3 phosphorylation (Tyr705). Furthermore, DSF/Cu treatment resulted in the downregulation of cyclin D1 and survivin expression, which are downstream targets of STAT3 signaling. Interestingly, phosphorylated STAT3 and STAT3 downstream signaling were highly upregulated in the mammospheres, and these upregulated molecules were significantly decreased by DSF/Cu treatment. In MDA-MB-231-derived xenograft model, DSF administration elicited a significant reduction in tumor growth with a noticeable downregulation of CD44, ALDH1 and phosphorylated STAT3 levels. Taken together, our results suggest that therapeutic treatment to DSF can effectively manifest antitumor activity by reducing CSC-like properties, and that STAT3 could be a key molecule of aggressive TNBC that is targeted by DSF. Note: This abstract was not presented at the meeting. Citation Format: Yoon-Jae Kim, Nahyun Lee, Eunhye Oh, Daeil Sung, Tae-Min Cho, Ji Young Kim, Jae Hong Seo. Disulfiram possesses antitumor activity and inhibits cancer stem-like properties via inhibition of STAT3 activation in triple-negative breast cancer cells [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 4302. doi:10.1158/1538-7445.AM2017-4302

Abstract 115: Flubendazole targets cancer stem-like properties and the HER2/Akt signaling pathway in HER2-positive breast cancer with trastuzumab resistance

Background and Propose: HER2-positive breast cancers are associated with an aggressive phenotype and trastuzumab resistance leading to poor clinical outcomes. Flubendazole (FLU) is a potent anthelmintic agent that inhibits microtubule polymerization, and also exhibits anticancer activity in several cancer types. The objective of the present study was to investigate the effect of FLU on apoptosis, HER2/Akt signaling pathway, cancer stem cell (CSC)-like properties and trastuzumab-resistance in HER2-positive breast cancer cells. Methods: FLU-induced antitumor effects were characterized by MTS assay, Sub-G1 and G2/M-phase arrest population, Annexin-V analysis, ALDH1 activity assay, Western blotting and immunofluorescence. For in vivo study, trastuzumab-resistant JIMT1 cells were injected into the mammary fat pads of BALB/c nude mice. We then determined the tumor growth and CSC-like properties. Results and conclusion: FLU significantly reduced cell viability and induced apoptosis with accompanied by activation of caspase-3, -7, and -8, as well as PARP cleavage in HER2-positive cell lines (MDA-MB-453, SKBR3 and BT474). FLU-induced apoptosis is associated with significant decreases in expression levels of HER2, p-HER2 (Tyr1221/1222), HER3, and p-HER3 (Tyr1289). FLU treatment also resulted in G2/M-phase arrest of the cell cycle, as evidenced by a marked downregulation of phospho-Histone H3 expression. These results were closely related to the decline of CSC-like properties, coinciding with the suppression of ALDH1 activity. These effects were also observed in trastuzumab-resistant JIMT-1 cells, and significant reductions in tumor growth and ALDH1A1 downregulation were observed in xenograft models together with downregulation of HER2 expression in vivo. Our findings suggest that FLU may be potentially effective for the treatment of HER2-positive breast cancer with trastuzumab resistance. Note: This abstract was not presented at the meeting. Citation Format: Daeil Sung, Youngkwan Cho, Eunhye Oh, Tae-Min Cho, Yoon-Jae Kim, Ji Young Kim, Jae hong Seo. Flubendazole targets cancer stem-like properties and the HER2/Akt signaling pathway in HER2-positive breast cancer with trastuzumab resistance [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 115. doi:10.1158/1538-7445.AM2017-115