Hyang Ju Lee

Radioresistant Cancer Cells Can Be Conditioned to Enter Senescence by mTOR Inhibition

Autophagy is frequently activated in radioresistant cancer cells where it provides a cell survival strategy. The mTOR inhibitor rapamycin activates autophagy but paradoxically it also enhances radiosensitivity. In this study, we investigated the mechanisms of these opposing actions in radiation-resistant glioma or parotid carcinoma cells. Radiation treatment transiently enhanced autophagic flux for a period of 72 hours in these cells and treatment with rapamycin or the mTOR inhibitor PP242 potentiated this effect. However, these treatments also increased heterochromatin formation, irreversible growth arrest, and premature senescence, as defined by expression of senescence-associated β-galactosidase activity. This augmentation in radiosensitivity seemed to result from a restoration in the activity of the tumor suppressor RB and a suppression of RB-mediated E2F target genes. In tumor xenografts, we showed that administering rapamycin delayed tumor regrowth after irradiation and increased senescence-associated β-galactosidase staining in the tumor. Our findings suggest that a potent and persistent activation of autophagy by mTOR inhibitors, even in cancer cells where autophagy is occurring, can trigger premature senescence as a method to restore radiosensitivity.

Senescence-Associated MCP-1 Secretion Is Dependent on a Decline in BMI1 in Human Mesenchymal Stromal Cells

AIMS Cellular senescence and its secretory phenotype (senescence-associated secretory phenotype [SASP]) develop after long-term expansion of mesenchymal stromal cells (MSCs). Further investigation of this phenotype is required to improve the therapeutic efficacy of MSC-based cell therapies. In this study, we show that positive feedback between SASP and inherent senescence processes plays a crucial role in the senescence of umbilical cord blood-derived MSCs (UCB-MSCs). RESULTS We found that monocyte chemoattractant protein-1 (MCP-1) was secreted as a dominant component of the SASP during expansion of UCB-MSCs and reinforced senescence via its cognate receptor chemokine (c-c motif) receptor 2 (CCR2) by activating the ROS-p38-MAPK-p53/p21 signaling cascade in both an autocrine and paracrine manner. The activated p53 in turn increased MCP-1 secretion, completing a feed-forward loop that triggered the senescence program in UCB-MSCs. Accordingly, knockdown of CCR2 in UCB-MSCs significantly improved their therapeutic ability to alleviate airway inflammation in an experimental allergic asthma model. Moreover, BMI1, a polycomb protein, repressed the expression of MCP-1 by binding to its regulatory elements. The reduction in BMI1 levels during UCB-MSC senescence altered the epigenetic status of MCP-1, including the loss of H2AK119Ub, and resulted in derepression of MCP-1. INNOVATION Our results provide the first evidence supporting the existence of the SASP as a causative contributor to UCB-MSC senescence and reveal a so far unappreciated link between epigenetic regulation and SASP for maintaining a stable senescent phenotype. CONCLUSION Senescence of UCB-MSCs is orchestrated by MCP-1, which is secreted as a major component of the SASP and is epigenetically regulated by BMI1.

Calpain Determines the Propensity of Adult Hippocampal Neural Stem Cells to Autophagic Cell Death Following Insulin Withdrawal.

Programmed cell death (PCD) has significant effects on the function of neural stem cells (NSCs) during brain development and degeneration. We have previously reported that adult rat hippocampal neural stem (HCN) cells underwent autophagic cell death (ACD) rather than apoptosis following insulin withdrawal despite their intact apoptotic capabilities. Here, we report a switch in the mode of cell death in HCN cells with calpain as a critical determinant. In HCN cells, calpain 1 expression was barely detectable while calpain 2 was predominant. Inhibition of calpain in insulin‐deprived HCN cells further augmented ACD. In contrast, expression of calpain 1 switched ACD to apoptosis. The proteasome inhibitor lactacystin blocked calpain 2 degradation and elevated the intracellular Ca2+ concentration. In combination, these effects potentiated calpain activity and converted the mode of cell death to apoptosis. Our results indicate that low calpain activity, due to absence of calpain 1 and degradation of calpain 2, results in a preference for ACD over apoptosis in insulin‐deprived HCN cells. On the other hand, conditions leading to high calpain activity completely switch the mode of cell death to apoptosis. This is the first report on the PCD mode switching mechanism in NSCs. The dynamic change in calpain activity through the proteasome‐mediated modulation of the calpain and intracellular Ca2+ levels may be the critical contributor to the demise of NSCs. Our findings provide a novel insight into the complex mechanisms interconnecting autophagy and apoptosis and their roles in the regulation of NSC death. Stem Cells 2015;33:3052—3064

Promoter methylation of WNT inhibitory factor-1 and expression pattern of WNT/β-catenin pathway in human astrocytoma: pathologic and prognostic correlations.

WNT inhibitory factor-1 (WIF1) is an antagonist of the WNT signaling pathway. We investigated the relationship between WIF1 promoter methylation and regulation of the WNT/β-catenin signaling pathway, tumor grade, and survival in patients with astrocytoma. This study included 86 cases of astrocytoma, comprising 20 diffuse astrocytomas and 66 glioblastomas. In addition, 17 temporal lobectomy specimens from patients with epilepsy were included as controls. The ratio of methylated DNA to total methylated and unmethylated DNA (% methylation) was measured by methylation- and unmethylation-specific PCR. Representative tumor tissue was immunostained for WIF1, β-catenin, cyclin D1, c-myc, and isocitrate dehydrogenase 1. Levels of WIF1 promoter methylation, mRNA expression, and protein expression in a glioblastoma cell line were compared before and after demethylation treatment. The mean percent methylation of the WIF1 promoter in astrocytomas was higher than that in control brain tissue. WIF1 protein expression was lower in the tumor group with >5% methylation than in the group with <5% methylation. Cytoplasmic β-catenin staining was more frequently observed in tumors with a low WIF1 protein expression level. Demethylation treatment of a glioblastoma cell line increased WIF1 mRNA and protein expression. Increased WIF1 promoter methylation and decreased WIF1 protein expression were not related to patient survival. In conclusion, WIF1 expression is downregulated by promoter methylation and is an important mechanism of aberrant WNT/β-catenin pathway activation in astrocytoma pathogenesis.

Elevated Pentraxin 3 in Obese Adipose Tissue Promotes Adipogenic Differentiation by Activating Neuropeptide Y Signaling

Obesity is accompanied by chronic systemic inflammation characterized by macrophage infiltration of obese tissues, an elevated plasma level of inflammatory substances, and excessive accumulation of lipids. The pro-inflammatory factor pentraxin 3 (PTX3) is also elevated in obese tissues, suggesting its potential role in adipogenesis. We found by analyzing murine preadipocyte 3T3-L1 cells, and human adipocytes derived from mesenchymal stem cells, which locally elevated PTX3 in obese adipose tissue augments adipocyte differentiation and subsequent lipid accumulation. This occurs via the upregulation of adipogenesis-related transcription factors. PTX3 enhanced lipid accumulation in murine 3T3-L1 cells by upregulating the expression of neuropeptide Y (NPY)/NPY receptor (NPYR) expression in preadipocytes. Pharmacological inhibition by NPYR antagonists abolished these effects. NPY also promoted the production of reactive oxygen species (ROS), a known trigger of adipogenesis. NPYR antagonists as well as antioxidant N-acetylcysteine showed anti-adipogenic effects by reducing the ROS levels, indicating that PTX3 mediates adipogenesis through NPY-dependent ROS production. These findings suggest that PTX3 plays a key role in the development of obesity by enhancing adipocyte differentiation and lipid synthesis via NPY/NPYR signaling. These observations provide a mechanistic explanation for the adipogenesis mediated by PTX3.

Abstract 144: Src leads to a novel mechanism of resistance to PI3K inhibitors through regulation of PI3K/p85 activation

Activation of the PI3K pathway is commonly observed and is correlated with tumor development, progression, poor prognosis, and resistance to cancer therapies, such as radiotherapy, in most cancers. As a central node of this pathway, PI3K is an attractive target for PI3K-addicted cancer therapy and PI3K inhibitors may thus restore sensitivity to other treatments when administered as part of combination regimens. Here, we found that PI3K/p85 was expressed predominantly in the radioresistant head and neck cancer cell line (HN31 cell line). And then, we investigated whether PI3K modulation was crucial for the development of novel treatment strategies for radioresistant cancer cell line. Interestingly, we found that head and neck cancer cell lines with PI3K/p85 activation showed the resistance to PI3K inhibitors and the resistance mechanism was associated with Src activation which is a member of a superfamily of membrane-associated nonreceptor protein tyrosine kinases. Src inhibitor improves the efficacy of PI3K inhibitor treatment through suppression of Src and PI3K/p85 activation in HN31 cell line. Collectively, our study highlights the role of p85 and Src activation in the resistance for PI3K inhibition and the potential clinical application of combination regimens of Src and PI3K inhibitors in head and neck cancers. This is the first investigation to analyze the role of Src in resistance to the PI3K inhibitors of head and neck cancer. As a consequence, a greater understanding of resistance mechanisms through our results will enable the rational design of combination regimens and sequential treatment algorithms to improve clinical outcomes. Citation Format: Gui Chul Kim, Hae Yun Nam, Hyang Ju Lee, Min Kyung Kim, Geun Hee Lee, Myung Woul Han, Seong Who KIM, Sang Yoon Kim. Src leads to a novel mechanism of resistance to PI3K inhibitors through regulation of PI3K/p85 activation [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 144. doi:10.1158/1538-7445.AM2017-144

Abstract P3-07-34: Predictive role of stromal tumor infiltrating lymphocytes (TILs) in patients with metastatic HER2-positive breast cancer (BC) treated with trastuzumab

Background: Prognostic significance of stromal TILs in metastatic BC has been suggested in various BC subtypes. However, predictive role of stromal TILs for the efficacy of trastuzumab has not been established in patients with HER2-positive BC. This study was performed to evaluate whether the stromal TILs are associated with the efficacy of trastuzumab in patients with metastatic HER2-positve BC. Method: Between June 2006 and March 2013, a total of 60 women with recurrent or metastatic HER2-positive BC treated with trastuzumab were included in this retrospective analysis. In these patients, trastuzumab was administered either as single agent or combination with taxanes. Stromal TILs were assessed using immunohistochemistry in surgical specimen (n=39, 65%) and biopsy specimen of metastatic lesion (n=21, 35%) by the academic pathologist (HJL). Primary endpoint of this study was progression-free survival (PFS), and secondary endpoints were response rate and overall survival (OS). Result: Median age was 54 year old (range, 36-76), and all patients had invasive ductal carcinoma. Hormone receptor was positive in 34 patients (57%) and 18 patients (30%) initially presented with metastatic disease. Nine patients (15%) received cytotoxic chemotherapy without trastuzumab before the administration of trastuzumab. Patients were grouped according to the TILs ( 10% [n=10]), and there was no significant difference in age (p=0.68), histologic grade (p=1.00), metastatic sites (p>0.05), and number of lines of chemotherapy before the administration of trastuzumab(p=0.33) among patients with low and high stromal TILs. High TILs were more common in hormone receptor (HR)-negative tumor compared with HR–positive tumor (31% vs 6%; p=0.02). In overall, median PFS and OS were 15.0 months (95% CI, 9.7-20.2) and 35.0 months (95% CI, 29.8-40.2), respectively. Median PFS in patients with high stromal TILs were numerically longer than that in those with low TILs (22.0 months [95% CI, 9.6-34.4] vs 14.0 months [95% CI, 9.6-18.4]; p=0.057). There was no difference in response rates (p=0.43) and OS (p=0.94) according to the stromal TILs. FcR genotype was not significantly correlated with objective response rate, PFS and OS. Conclusion: This study suggests that the stromal TILs might be associated with the clinical outcomes of HER2-targeted therapy in patients with metastatic HER2-positive BC. Our finding should be validated in future studies based on a large sample size. Keywords: Breast cancer, tumor infiltrating lymphocyte. Trastzumab, HER2. Citation Format: Yoon JA, Yoo C, Lee HJ, Kim K-P, Kim J, Ahn J-H, Jung KH, Gong G, Kim S-B. Predictive role of stromal tumor infiltrating lymphocytes (TILs) in patients with metastatic HER2-positive breast cancer (BC) treated with trastuzumab. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-34.

Abstract P3-07-56: Predictive value of tertiary lymphoid structure assessed by high endothelial venule count in neoadjuvant setting of triple-negative breast cancer

Background Tertiary lymphoid structure (TLS) is an ectopic lymph node-like structure characterized by lymphoid aggregation with high endothelial venules (HEVs), and is an important source of tumor-infiltrating lymphocytes (TILs). TILs have a strong prognostic and predictive significance, particularly in triple-negative breast cancer (TNBC). We previously analyzed expression of immune-related genes in pre-neoadjuvant chemotherapy (NAC) biopsy samples using NanoString assay and showed that gene expression of follicular helper T cell marker CXCL13 , which is closely associated with TLSs, was an independent predictive factor for pathologic complete remission (pCR) in TNBC. However, measuring gene expression of biopsy sample is not easy to perform in daily pathology practice. Therefore, we evaluated TLSs by assessing hematoxylin and eosin (HE San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-56.

Abstract 4904: β-catenin silencing enhances radiation sensitivity through antagonizing effect of AMPK against Ku70/80 in head and neck cancer cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The Wnt/β-catenin pathway regulates the cell growth and survival following radiation in various types of cancer cells. Our previous report show that activation of the Wnt/β-catenin signaling pathway is a key radioprotective mechanism in irradiated head and neck cancer (HNC) cells. However, the molecular mechanisms by which β-catenin regulates radiation sensitivity are not clear. Here we attempted to elucidate the mechanism of cell death following radiation by studying how β-catenin silencing controls the radiation sensitivity of radioresistant HNC cells. Of nine cell lines examined, the most radioresistant cell line (AMC-HN-9) were selected for this experiments. β-catenin silencing using small interfering RNA(siRNA) down-regulated β-catenin expression up to 72 h, which was confirmed by western blot analysis. The sensitivity to radiation was anlayzed by clonogenic analysis and MTT assay. As a result, β-catenin silencing remarkably decreased the survival of irradiated AMC-HN-9 cells and the cell viability also significantly reduced more by the combination treatment with β-catenin siRNA and radiation (0.37±0.034 fold) than when treated with β-catenin siRNA or radiation alone (0.68±0.055 fold and 0.90±0.043 fold, respectively). Interestingly, whereas expression of Ku70/80 was up regulated in AMC-HN-9 cells following irradiation (4Gy), in the cells treated with combination of radiation and β-catenin siRNA, Ku70/80 expression was dramatically decreased. In additionally, when exposed to radiation after β-catenin silencing, the up-regulation of irradiation-induced Ku80 completely was prevented by β-catenin silencing-induced AMPK. Taken together, these results suggest that suppression of Ku70/80 expression through β-catenin silencing-induced AMPK is associated with its radio-sentitizing effect in AMC-HN-9 cells, thus supporting a novel radiosensitive mechanism of radioresistant HNC cells. Citation Format: Hyo Won Chang, Hae Yun Nam, Mi Ra Kim, Hyang Ju Lee, Ji Hyun Seo, So Yeon Lee, Seong Who Kim, Sang Yoon Kim. β-catenin silencing enhances radiation sensitivity through antagonizing effect of AMPK against Ku70/80 in head and neck cancer cells. [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 4904. doi:10.1158/1538-7445.AM2014-4904

Abstract P2-02-02: Prognostic significance of the ratio of absolute neutrophil to lymphocyte counts for breast cancer patients in neoadjuvant setting

Background: The baseline absolute neutrophil count/absolute lymphocyte count ratio (NLR) has potential prognostic importance in solid tumors, including breast cancer. However, NLR as a predictive factor of the pathologic complete response (pCR) or as a prognostic factor has not been fully studied in breast cancer patients who have received neoadjuvant chemotherapy. Materials and Methods: Using receiver operating characteristic curve analyses, we retrospectively examined the predictive and prognostic impact of the NLR at the diagnosis in 401 patients with primary breast cancer who were treated with neoadjuvant chemotherapy, followed by definitive surgical resection. Of the 401 included patients, 72 (18%) received anthracycline-based, 285 (71%) received anthracycline- and taxane-based, 37 (9.2%) received herceptin-based, and 7 (1.7%) received fluorouracil-based regimens. The significance of NLR was analyzed according to clinicopathologic variables and clinical outcomes. Results: Forty-three (10.7%) patients achieved pCR. On univariate analysis, high NLR (> 2.2) correlated with poor recurrence-free survival (RFS; P = 0.001) and overall survival (OS; P = 0.003). Subgroup analysis of the non-pCR group showed that NLR was significant for RFS and OS ( P = 0.002 and P = 0.007, respectively). Multivariate analysis showed NLR to be an independent prognostic factor for RFS and OS ( P = 0.002 and P = 0.007, respectively). Subgroup analysis of the pCR group showed that patients with high NLR have a higher recurrent rate than low NLR (30.8% vs. 13.3%). The NLR was not related to pCR ( P = 0.098). Conclusion: Our results suggest that the NLR is an independent prognostic factor for RFS and OS in breast cancer patients receiving neoadjuvant chemotherapy and may provide additional prognostic information for patients not achieving a pCR to determine who might profit from further therapy. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-02-02.