The monocyte chemoattractant protein-1 (MCP-1)/CCR2 system is involved in peritoneal dialysis-related epithelial-mesenchymal transition of peritoneal mesothelial cells.

Lee, Sun Ha,Kang, Hye-Young,Kim, Kyung Sik,Nam, Bo Young,Paeng, Jisun,Kim, Seonghun,Li, Jin Ji,Park, Jung Tak,Kim, Dong Ki,Han, Seung Hyeok,Yoo, Tae-Hyun,Kang, Shin-Wook United States and Canadian Academy of Pathology [e 2012 Laboratory investigation Vol.92 No.12

<P>Epithelial-mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs) has a role in the process of peritoneal fibrosis (PF), a serious complication in peritoneal dialysis (PD) patients. Even though monocyte chemoattractant protein-1 (MCP-1) was demonstrated to directly increase extracellular matrix (ECM) synthesis, the role of the MCP-1/CCR2 system in PD-related EMT and ECM synthesis in cultured human PMCs (HPMCs) and in an animal model of PD has never been elucidated. In vitro, HPMCs were exposed to 5.6 mM glucose (NG), NG+MCP-1 (10?ng/ml) (NG+MCP-1), or 100?mM glucose (HG) with or without CCR2 inhibitor (RS102895) (CCR2i) or a dominant-negative mutant MCP-1-expressing lentivirus (LV-mMCP-1). In vivo, PD catheters were inserted into 60 Sprague-Dawley rats, and saline (Control, C) (N=30) or 4.25% PD solution (PD) (N=30) was infused for 4 weeks. Twenty rats from each group were treated with empty LV or LV-mMCP-1 intraperitoneally. Snail, E-cadherin, α-smooth muscle actin (α-SMA), and fibronectin protein expression in HPMCs and the peritoneum was evaluated by western blot analysis. Compared with NG cells, Snail, α-SMA, and fibronectin expression was significantly increased, while E-cadherin expression was significantly decreased in HPMCs exposed to HG and NG+MCP-1, and these changes were significantly abrogated by CCR2i (P<0.05). In addition, MCP-1-induced EMT was significantly attenuated by anti-TGF-β1 antibody. In PD rats, Snail and fibronectin expression was significantly increased in the peritoneum, whereas the ratios of E-cadherin/α-SMA protein expression were significantly decreased (P<0.05). The thickness of the peritoneum and the intensity of Masson's trichrome staining in the peritoneum were also significantly higher in PD rats than in C rats (P<0.05). These changes in PD rats were significantly abrogated by LV-mMCP-1. These findings suggest that the MCP-1/CCR2 system is directly involved in PD-related EMT and ECM synthesis and that this is mediated, at least in part, via TGF-β1.</P>

Elevated TRAF2/6 expression in Parkinson's disease is caused by the loss of Parkin E3 ligase activity.

Chung, Ji-Yun,Park, Hee Ra,Lee, Su-Jin,Lee, Sun-Hye,Kim, Jin Sik,Jung, Youn-Sang,Hwang, Sang Hyun,Ha, Nam-Chul,Seol, Won-Gi,Lee, Jaewon,Park, Bum-Joon United States and Canadian Academy of Pathology [e 2013 Laboratory investigation Vol.93 No.6

<P>Parkinson's disease (PD) is the second leading neurodegenerative disease, and is known to be induced by environmental factors or genetic mutations. Among the verified genetic mutations of PD, Parkin, isolated from the PARK2 locus, shows an autosomal recessive inheritance pattern and is known to be an E3 ligase. However, the physiological target of Parkin and the molecular mechanism of Parkin-deficiency-induced PD have not been clearly demonstrated until now. It has recently been proposed that inflammation, suggesting as a causal factor for PD, is enhanced by Parkin deficiency. Thus, we examined the relationship between inflammation-related factors and Parkin. Here, we provide the evidence that Parkin suppresses inflammation and cytokine-induced cell death by promoting the proteasomal degradation of TRAF2/6 (TNF-α receptor-associated factor 2/6). Overexpression of Parkin can reduce the half-lives of TRAF2 and TRAF6, whereas si-Parkin can extend them. However, mutant Parkins did not alter the expression of TRAF2/6. Thus, loss of Parkin enhances sensitivity to TNF-α- or IL-1β-induced JNK activation and NF-κB activation. Indeed, si-Parkin-induced apoptosis is suppressed by the knockdown of TRAF6 or TRAF2. We also observed elevated expression levels of TRAF6 and a reduction of IκB in an 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced PD mouse model. Moreover, elevated expression levels or aggregation of TRAF6 were detected in approximately half of the human PD tissues (7/15 cases) and 2 cases, respectively. In addition, TRAF6 and Parkin expression levels show a reverse relationship in human PD tissues. Our results strongly suggest that the reduction of Parkin or overexpression of TRAF2/6 by chronic inflammation would be the reason for occurrence of PD.</P>

Effects of dexamethasone on the TGF-β1-induced epithelial-to-mesenchymal transition in human peritoneal mesothelial cells.

Jang, Yang-Hee,Shin, Hyun-Soo,Sun Choi, Hack,Ryu, Eun-Sun,Jin Kim, Mi,Ki Min, Seog,Lee, Joo-Ho,Kook Lee, Hyeon,Kim, Kwang-Ho,Kang, Duk-Hee United States and Canadian Academy of Pathology [e 2013 Laboratory investigation Vol.93 No.2

<P>The epithelial-to-mesenchymal transition (EMT) is known to have a role in appropriate embryonic development, the physiological response to injury and pathological events such as organ fibrosis and cancer progression. Glucocorticoid (GC), one of the most commonly used anti-inflammatory drugs, inhibits the deposition of extracellular matrix independent of its anti-inflammatory effect. The EMT of human peritoneal mesothelial cells (HPMCs) is a key mechanism of peritoneal fibrosis; however, it has not yet been investigated whether GC imposes any effect on the EMT of HPMCs. To investigate the therapeutic potential of GC on preserving peritoneal membrane function, we studied the effect of dexamethasone (DEXA), a synthetic GC, on the transforming growth factor-β1 (TGF-β1)-induced EMT in HPMCs. As assessed by changes in cell morphology, the expression of epithelial and mesenchymal cell markers (such as E-cadherin, ZO-1 and α-SMA, α-smooth muscle actin) and cell migration, DEXA inhibited the TGF-β1-induced EMT. RU486, a glucocorticoid receptor (GR) antagonist, blocked the effect of DEXA on the TGF-β1-induced EMT. Importantly, DEXA also induced the mesenchymal-to-epithelial transition of TGF-β1-stimulated HPMCs. The beneficial effect of DEXA on the TGF-β1-induced EMT was mediated through the amelioration of ERK and p38 mitogen-activated protein kinase (MAPK) phosphorylation; however, this effect was not related to the TGF-β1-induced activation of Smad2/3 signaling. DEXA inhibited glycogen synthase kinase-3β (GSK-3β) phosphorylation and the Snail upregulation induced by TGF-β1, which were also ameliorated by inhibitors of MAPK. In conclusion, this is the first study demonstrating the protective effect of DEXA on the EMT in TGF-β1-stimulated HPMCs by inhibiting MAPK activation, GSK-3β phosphorylation and Snail upregulation.</P>

Heat shock protein 90 inhibitor attenuates renal fibrosis through degradation of transforming growth factor-관 type II receptor.

Noh, Hyunjin,Kim, Hyun J,Yu, Mi R,Kim, Wan-Young,Kim, Jin,Ryu, Jung H,Kwon, Soon H,Jeon, Jin S,Han, Dong C,Ziyadeh, Fuad United States and Canadian Academy of Pathology [e 2012 Laboratory investigation Vol.92 No.11

<P>The accumulation of extracellular matrix proteins in the interstitial area is the final common feature of chronic kidney diseases. Accumulating evidence suggests that transforming growth factor (TGF)-관1 promotes the development of renal fibrosis. Heat shock protein (Hsp) 90 inhibitors have been shown to repress TGF-관1 signaling, but whether they inhibit renal fibrosis is unknown. The purpose of this study is to determine the therapeutic efficacy of Hsp90 inhibitor on renal fibrosis. In TGF-관1-treated HK2 cells and unilateral ureteral obstruction (UUO) kidneys, we found that 17-allylamino-17-demethoxygeldanamycin (17AAG), an Hsp90 inhibitor, decreased the expression of 관-smooth muscle actin, fibronectin, and collagen I and largely restored the expression of E-cadherin. 17AAG inhibited TGF-관1-mediated phosphorylation of Smad2, Akt, glycogen synthase kinase-3관, and extracellular signal-regulated kinase in HK2 cells. Inhibition of Hsp90 also blocked TGF-관1-mediated induction of snail1. This 17AAG-induced reduction was completely restored by simultaneous treatment with proteasome inhibitor MG132. Furthermore, 17AAG blocked the interaction between Hsp90 and TGF-관 type II receptor (T관RII) and promoted ubiquitination of T관RII, leading to the decreased availability of T관RII. Smurf2-specific siRNA reversed the ability of 17AAG to inhibit TGF-관1 signaling. The effect of 17AAG on T관RII expression and renal fibrosis was confirmed in UUO kidneys. These findings suggest that Hsp90 inhibitor prevents the development of renal fibrosis via a mechanism dependent on Smurf2-mediated degradation of T관RII.</P>

Indoxyl sulfate-induced epithelial-to-mesenchymal transition and apoptosis of renal tubular cells as novel mechanisms of progression of renal disease.

Kim, Su Hyun,Yu, Min-A,Ryu, Eun Sun,Jang, Yang-Hee,Kang, Duk-Hee United States and Canadian Academy of Pathology [e 2012 Laboratory investigation Vol.92 No.4

<P>Indoxyl sulfate (IS), one of the uremic toxins, is regarded to have a substantial role in the progression of chronic kidney disease (CKD). Epithelial-to-mesenchymal transition (EMT) and apoptosis of renal tubular cells are known to be the critical mechanisms of the development and aggravation of CKD. We investigated the effect of IS on EMT and apoptosis in renal proximal tubular cells, NRK-52E cells. IS significantly inhibited cell proliferation and induced cell migration with a morphological transition from cuboidal epithelial cells to spindle-shaped scattered fibroblast-like cells. IS downregulated the expressions of zonula occluden-1 and E-cadherin, whereas upregulated α-SMA expression at 48?h, which was blocked by a pretreatment of the organic anion transporter, probenecid. IS also induced apoptosis of NRK cells from a concentration of 25?μg/ml with an activation of ERK1/2 and p38 MAP kinase (MAPK). Pretreatment of ERK1/2 or p38 MAPK inhibitors, PD98059 or SB203580, resulted in no significant effect on IS-induced EMT, whereas it ameliorated IS-induced apoptosis of NRK cells. These findings suggested phenotypic transition and apoptosis as potential mechanisms of IS-induced renal damage and the differential role of MAPK activation in IS-induced EMT and apoptosis of renal tubular cells.</P>

Non-histone nuclear factor HMGB1 is phosphorylated and secreted in colon cancers.

Kang, Hyun Ju,Lee, Hanna,Choi, Hee-Jung,Youn, Ju Ho,Shin, Jeon-Soo,Ahn, Yeong Hee,Yoo, Jong Shin,Paik, Young-Ki,Kim, Hoguen United States and Canadian Academy of Pathology [e 2009 Laboratory investigation Vol.89 No.8

<P>The high mobility group box 1 (HMGB1) protein, a non-histone nuclear factor, is overexpressed and localizes to the cytoplasm in some cancer cells. However, the mechanism of cytoplasmic HMGB1 transport, extracellular secretion, and its role in cancer progression is not clear. To simulate the activated state of HMGB1, we mutated serine residues of nuclear localization signals (NLSs) to glutamic acid and performed transfection assays. We carried out a kinase inhibitor study and evaluated the cell migration by invasion assay. We showed that phosphorylated HMGB1 localizes in the cytoplasm of colon cancer cells and also showed the interaction of PKC and HMGB1 by immunoprecipitation analysis. Concurrent mutations at six serine residues (35, 39, 42, 46, 53, and 181) to glutamic acid induced the nuclear to cytoplasmic transport of HMGB1, which was detected in the culture medium. We also observed that the secretion of HMGB1 correlated with increased cancer cell invasiveness. Our results suggest that phosphorylated HMGB1 is transported to the cytoplasm, is subsequently secreted from the cell, and has a role in tumor progression through the activation of genes related to cell migration.</P>

The role of translation elongation factor eEF1A in intracellular alkalinization-induced tumor cell growth.

Kim, Juno,Namkung, Wan,Yoon, Jae Seok,Jo, Min Jae,Lee, Sung Hee,Kim, Kyung Hwan,Kim, Joo Young,Lee, Min Goo United States and Canadian Academy of Pathology [e 2009 Laboratory investigation Vol.89 No.8

<P>The formation of a pH gradient, which is characterized by intracellular alkalinization and extracellular acidification, plays a key role in the growth and metastasis of tumor cells. However, the underlying mechanisms of alkalinization-induced cell growth are not known. In this study, we investigated the roles of eukaryotic translation elongation factor 1 alpha (eEF1A) in alkalinization-induced cell growth. In all cell lines tested (NIH3T3, HEK293, and HeLa), cell growth was affected by the modulation of intracellular pH. In general, weak intracellular alkalinization produced increased cell growth, whereas intracellular acidification resulted in decreased cell growth. It is interesting to note that portions of actin-bound eEF1A proteins were gradually reduced from acidic to alkaline conditions, suggesting an increase in levels of functionally active, free-form eEF1A. Over-expression of eEF1A caused increased cell growth in HeLa cells. It should be noted that dissociation of eEF1A from actin by transfection with the actin-binding domain deleted eEF1A construct further increased cell growth under acidic conditions, whereas most of the intact eEF1A was bound to actin. Conversely, knockdown of eEF1A by treatment with eEF1A1 and eEF1A2 siRNAs nullified the effects of alkalinization-induced cell growth. The above findings suggest that an increase in free-form eEF1A under alkaline conditions plays a critical role in alkalinization-induced cell growth.</P>

Clinical and biological implications of CD133-positive and CD133-negative cells in glioblastomas.

Joo, Kyeung Min,Kim, Shi Yean,Jin, Xun,Song, Sang Yong,Kong, Doo-Sik,Lee, Jung-Ii,Jeon, Ji Won,Kim, Mi Hyun,Kang, Bong Gu,Jung, Yong,Jin, Juyoun,Hong, Seung-Chyul,Park, Woong-Yang,Lee, Dong-Sup,Kim, H United States and Canadian Academy of Pathology [e 2008 Laboratory investigation Vol.88 No.8

<P>A number of recent reports have demonstrated that only CD133-positive cancer cells of glioblastoma multiforme (GBM) have tumor-initiating potential. These findings raise an attractive hypothesis that GBMs can be cured by eradicating CD133-positive cancer stem cells (CSCs), which are a small portion of GBM cells. However, as GBMs are known to possess various genetic alterations, GBMs might harbor heterogeneous CSCs with different genetic alterations. Here, we compared the clinical characteristics of two GBM patient groups divided according to CD133-positive cell ratios. The CD133-low GBMs showed more invasive growth and gene expression profiles characteristic of mesenchymal or proliferative subtypes, whereas the CD133-high GBMs showed features of cortical and well-demarcated tumors and gene expressions typical of proneuronal subtype. Both CD133-positive and CD133-negative cells purified from four out of six GBM patients produced typical GBM tumor masses in NOD-SCID brains, whereas brain mass from CD133-negative cells showed more proliferative and angiogenic features compared to that from CD133-positive cells. Our results suggest, in contrast to previous reports that only CD133-positive cells of GBMs can initiate tumor formation in vivo CD133-negative cells also possess tumor-initiating potential, which is indicative of complexity in the identification of cancer cells for therapeutic targeting.</P>

Radiosensitization of brain metastasis by targeting c-MET.

Yang, Heekyoung,Lee, Hye Won,Kim, Yonghyun,Lee, Yeri,Choi, Yeon-Sook,Kim, Kang Ho,Jin, Juyoun,Lee, Jeongwu,Joo, Kyeung Min,Nam, Do-Hyun United States and Canadian Academy of Pathology [e 2013 Laboratory investigation Vol.93 No.3

<P>Radiotherapy is the most widely used therapeutic modality in brain metastasis; however, it only provides palliation due to inevitable tumor recurrence. Resistance of tumor cells to ionizing radiation is a major cause of treatment failure. A critical unmet need in oncology is to develop rationale driven approaches that can enhance the efficacy of radiotherapy against metastatic tumor. Utilizing in vivo orthotopic primary tumor and brain metastasis models that recapitulate clinical situation of the patients with metastatic breast cancer, we investigated a molecular mechanism through which metastatic tumor cells acquire resistance to radiation. Recent studies have demonstrated that the hepatocyte growth factor (HGF)-c-Met pathway is essential for the pathologic development and progression of many human cancers such as proliferation, invasion and resistance to anticancer therapies. In this study, c-Met signaling activity as well as total c-Met expression was significantly upregulated in both breast cancer cell lines irradiated in vitro and ex vivo radio-resistant cells derived from breast cancer brain metastatic xenografts. To interrogate the role of c-Met signaling in radioresistance of brain metastasis, we evaluated the effects on tumor cell viability, clonogenicity, sensitivity to radiation, and in vitro/in vivo tumor growth after targeting c-Met by small-hairpin RNA (shRNA) or small-molecule kinase inhibitor (PF-2341066). Although c-Met silencing or radiation alone demonstrated a modest decrease in clonogenic growth of parental breast cancers and brain metastatic derivatives, combination of two modalities showed synergistic antitumor effects resulting in significant prolongation of overall survival in tumor-bearing mice. Taken together, optimizing c-Met targeting in combination with radiation is critical to enhance the effectiveness of radiotherapy in the treatments of brain metastasis.</P>

miR-106b modulates cancer stem cell characteristics through TGF-β/Smad signaling in CD44-positive gastric cancer cells.

Yu, Dayeon,Shin, Hyun-Soo,Lee, Yeo Song,Lee, Yong Chan United States and Canadian Academy of Pathology [e 2014 Laboratory investigation Vol.94 No.12

<P>Cancer stem cells have the capacity to form new tumors and are thus considered to be a cause of metastasis and tumor recurrence. However, many of the mechanisms determining cancer stem cell characteristics are still unknown. MicroRNAs (miRNAs) are possible modulators of cancer stem cell generation and may be involved in the retention of cancer stem cell characteristics. The aim of this study was to examine the miRNA expression profiles regulating the cancer stem-like cell characteristics in gastric cancer. We sorted gastric cancer stem-like cells using the stem cell marker CD44 by fluorescence-activated cell sorting. CD44(+) cells formed more and larger spheres compared with CD44(-) cells. Cancer stem cell markers were overexpressed in CD44(+) cells. CD44(+) cells showed increased expression of mesenchymal cell markers, whereas epithelial markers were downregulated. In miRNA microarray, the miR-106b family comprising miR-106b, miR-93, and miR-25 was significantly upregulated in CD44(+) cells than in CD44(-) cells. Smad7, which inhibits transforming growth factor-β (TGF-β)/Smad signaling as a target of the miR-106b family, was downregulated in CD44(+) cells. Furthermore, expression of TGF-β/Smad signal molecules was activated in CD44(+) cells, in accordance with the action of the miR-106b family. Inhibition of miR-106b showed suppression of the TGF-β/Smad signaling pathway and decreased self-renewal capacity and cell invasiveness. Our study suggests that CD44(+) gastric cancer cells show cancer stem cell properties with epithelial-mesenchymal transition (EMT). Increased miR-106b family expression regulated cancer stem-like cell properties, particularly EMT characteristics, through the TGF-β/Smad signaling pathway in CD44(+) stem-like cells. Taken together, these results indicate that targeting miR-106b may be an effective form of cancer therapy in gastric cancer through the modulation of cancer stem cell characteristics.</P>