Paradoxical Regulation of Hypoxia Inducible Factor-1α (HIF-1α) by Histone Deacetylase Inhibitor in Diffuse Large B-Cell Lymphoma

Hypoxia inducible factor (HIF) is important in cancer, as it regulates various oncogenic genes as well as genes involved in cell survival, proliferation, and migration. Elevated HIF-1 protein promotes a more aggressive tumor phenotype, and greater HIF-1 expression has been demonstrated to correlate with poorer prognosis, increased risk of metastasis and increased mortality. Recent reports suggest that HIF-1 activates autophagy, a lysosomal degradation pathway which may promote tumor cell survival. We show here that HIF-1α expression is constitutively active in multiple diffuse large B cell lymphoma (DLBCL) cell lines under normoxia and it is regulated by the PI3K/AKT pathway. PCI-24781, a pan histone deacetylase inhibitor (HDACI), enhanced accumulation of HIF-1α and induced autophagy initially, while extended incubation with the drug resulted in inhibition of HIF-1α. We tested the hypothesis that PCI-24781- induced autophagy is mediated by HIF-1α and that inhibition of HIF-1α in these cells results in attenuation of autophagy and decreased survival. We also provide evidence that autophagy serves as a survival pathway in DLBCL cells treated with PCI-24781 which suggests that the use of autophagy inhibitors such as chloroquine or 3-methyl adenine in combination with PCI-24781 may enhance apoptosis in lymphoma cells.     

RuvBL2 Is Involved in Histone Deacetylase Inhibitor PCI-24781-Induced Cell Death in SK-N-DZ Neuroblastoma Cells

Neuroblastoma is the second most common solid tumor diagnosed during infancy. The survival rate among children with high-risk neuroblastoma is less than 40%, highlighting the urgent needs for new treatment strategies. PCI-24781 is a novel hydroxamic acid-based histone deacetylase (HDAC) inhibitor that has high efficacy and safety for cancer treatment. However, the underlying mechanisms of PCI-24781 are not clearly elucidated in neuroblastoma cells. In the present study, we demonstrated that PCI-24781 treatment significantly inhibited tumor growth at very low doses in neuroblastoma cells SK-N-DZ, not in normal cell line HS-68. However, PCI-24781 caused the accumulation of acetylated histone H3 both in SK-N-DZ and HS-68 cell line. Treatment of SK-N-DZ with PCI-24781 also induced cell cycle arrest in G2/M phase and activated apoptosis signaling pathways via the up-regulation of DR4, p21, p53 and caspase 3. Further proteomic analysis revealed differential protein expression profiles between non-treated and PCI-24781 treated SK-N-DZ cells. Totally 42 differentially expressed proteins were identified by MALDI-TOF MS system. Western blotting confirmed the expression level of five candidate proteins including prohibitin, hHR23a, RuvBL2, TRAP1 and PDCD6IP. Selective knockdown of RuvBL2 rescued cells from PCI-24781-induced cell death, implying that RuvBL2 might play an important role in anti-tumor activity of PCI-24781 in SK-N-DZ cells. The present results provide a new insight into the potential mechanism of PCI-24781 in SK-N-DZ cell line.     

Suppression of urokinase plasminogen activator receptor inhibits proliferation and migration of pancreatic adenocarcinoma cells via regulation of ERK/p38 signaling

Pancreatic ductal adenocarcinoma (PDAC) expresses high levels of urokinase-type plasminogen activator (uPA), its receptor (uPAR) and plasminogen activator inhibitor (PAI)-2, which may play an important role in PDAC progression. The overexpression of uPAR predicted short survival in PDAC patients. In this study, two different PDAC cell lines were used to examine the effect of small interfering (si) RNAs to uPAR, uPA and PAI-2 on proliferation, apoptosis, migration and MAP kinase activation. In both PDAC cell lines, siRNA to uPAR significantly inhibited cell proliferation and migration and stimulated apoptosis, to a greater extent than uPA siRNA. When either PDAC cell line was treated with uPAR siRNA, the level of phosphorylated ERK (p-ERK) decreased substantially, whereas phosphorylated p38 (p-p38) increased when compared to non-silencing control, uPA siRNA or PAI-2 siRNA treatment. This resulted in enhancement of the p-p38/p-ERK ratio which favors cancer cell arrest. Interestingly, uPAR protein expression was suppressed by p-ERK inhibition and stimulated with p-p38 inhibition, suggesting the presence of a positive feedback loop between uPAR and ERK. In summary, our data indicate that, of the uPA system, uPAR exerts the strongest effects on PDAC cells, by acting through the ERK signaling pathway via a positive feedback loop. Disruption of this loop with uPAR siRNA or inhibitor of p-ERK, inhibits PDAC proliferation and migration and promotes apoptosis. These findings suggest that uPAR strongly contributes to PDAC progression and may be considered as a potential anti-pancreatic cancer target.     

GBP3 promotes glioma cell proliferation via SQSTM1/p62-ERK1/2 axis

Guanylate binding proteins (GBPs) are interferon-inducible large GTPases and play a crucial role in cell-autonomous immunity. However, the biology function of GBPs in cancer remains elusive. GBP3 is specifically expressed in adult brain. Here we show that GBP3 is highly elevated in human glioma tumors and glioma cell lines. Overexpression of GBP3 dramatically increased glioma cell proliferation whereas silencing GBP3 by RNA interference produced opposite effects. We further showed that GBP3 expression was able to induce sequestosome-1(SQSTM1, also named p62) expression and activate extracellular signal-regulated kinase (ERK1/2). The SQSTM1-ERK1/2 signaling cascade was essential for GBP3-promoted cell growth because depletion of SQSTM1 markedly reduced the phosphorylated ERK1/2 levels and GBP3-mediated cell growth, and inhibition of mitogen-activated protein kinase/ERK kinase abolished GBP3-induced glioma cell proliferation. Consistently, GBP3 overexpression significantly promoted glioma tumor growth in vivo and its expression was inversely correlated with the survival rate of glioma patients. Taken together, these results for the first time suggest that GBP3 contributes to the proliferation of glioma cells via regulating SQSTM1-ERK1/2 pathway, and GBP3 might represent as a new potential therapeutic target against glioma.     

Retracted: MicroRNA-99b suppresses human cervical cancer cell activity by inhibiting the PI3K/AKT/mTOR signaling pathway

Cervical cancer is common cancer among women with high morbidity. MicroRNAs (miRs) are involved in the progression and development of cervical cancer. This study aimed to explore the effect of miR-99b-5p (miR-99b) on invasion and migration in cervical cancer through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) signaling pathway. The microarray-based analysis was used to screen out differentially expressed miRNAs. Expression of miR-99b, PI3K, AKT, mTOR, and ribosomal protein S6 kinase (p70S6K) was determined in both cervical cancer tissues and paracancerous tissues. Next, alteration of miR-99b expression in cervical cancer was conducted to evaluate levels of PI3K, AKT, mTOR, p70S6K matrix metallopeptidase 2, epithelial cell adhesion molecule, and intercellular adhesion molecule 1, as well as the effect of miR-99b on cell proliferation, invasion, migration, cell cycle distribution, and apoptosis. The results demonstrated that miR-99b expression was decreased and levels of PI3K, AKT, mTOR, and p70S6K were elevated in cervical cancer tissues. More important, overexpressed miR-99b repressed the PI3K/AKT/mTOR signaling pathway, inhibited cell proliferation, invasion, and migration, blocked cell cycle entry, and promoted apoptosis in cervical cancer. These results indicate that miR-99b attenuates the migration and invasion of human cervical cancer cells through downregulation of the PI3K/AKT/mTOR signaling pathway, which provides a therapeutic approach for cervical cancer treatment.     

NUSAP1 knockdown inhibits cell growth and metastasis of non-small-cell lung cancer through regulating BTG2/PI3K/Akt signaling

Non-small-cell lung cancer (NSCLC) is the most common malignancy along with high mortality rate worldwide. Recently, nucleolar and spindle-associated protein 1 (NUSAP1) has been reported to be involved in the malignant progression of several cancers. However, in NSCLC, the biological function of NUSAP1 and its molecular mechanism have not been reported. Here, our findings indicated that the NUSAP1 messenger RNA expression level was remarkably upregulated in NSCLC tissues compared with that of adjacent normal tissues. We also found that NUSAP1 gene expression was notably upregulated in NSCLC cell lines (A549, 95-D, H358, and H1299) compared with that of normal human bronchial epithelial cell line (16HBE). Subsequently, the biological function of NUSAP1 was investigated in A549 and H358 cells transfected with NUSAP1 small interfering RNA (siRNA), respectively. Results showed that NUSAP1 knockdown inhibited NSCLC cell proliferation, and promoted cell apoptosis. Furthermore, the number of cell migration and invasion was significantly suppressed by NUSAP1 knockdown. In addition, our results indicated that NUSAP1 knockdown increased the gene expression of B-cell translocation gene 2 (BTG2), but decreased the expression levels of phosphoinositide 3-kinase (PI3K) and phosphorylated serine/threonine kinase (p-AKT). BTG2 siRNA partly abrogates the effect of NUSAP1 knockdown on BTG2 gene expression. Fumonisin B1 (FB1), a AKT activator, reversed the effect of NUSAP1 knockdown on the biological function in NSCLC. Taken together, NUSAP1 knockdown promotes NSCLC cell apoptosis, and inhibits cell proliferation, cell migration, and invasion, which is associated with regulating BTG2/PI3K/Akt signal pathway. Our findings suggest that NUSAP1 is a promising molecular target for NSCLC treatment.     

Retracted: Sapylin inhibits lung cancer cell proliferation and promotes apoptosis by attenuating PI3K/AKT signaling

Sapylin (OK-432) revealed biological properties in cancers. In this study, the effect of sapylin on lung cancer cell A549 was investigated. A549 cell lines were treated with sapylin (0.1, 0.5, and 1 KE/mL) for different time intervals. A549 cell proliferation and apoptosis was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide/Ki67 assay and flow cytometry, respectively. Western blot was used to determine the expressions of proteins involved in proliferation, apoptosis, and phosphoinositide 3-kinase/serine/threonine kinase (PI3K/AKT), Wnt3a/β-catenin signaling pathway. Level of intracellular reactive oxygen species (ROS) was insured by using the ROS kit. Sapylin inhibited A549 cell viability and the expressions of proliferation-related proteins (cyclin E1 and D1) in dose- and time-dependent manners. Sapylin promoted apoptosis in a dose- and time-dependent manners. Sapylin also promoted the expressions of apoptotic proteins (cleaved caspase-3 and 8) in dose- and time-dependent manners. Furthermore, sapylin increased the intracellular concentration of ROS in a dose-dependent manner. Besides, the high expression of ROS level might induce inhibition of cell viability and increase cell apoptosis. The mechanistic study revealed that sapylin inactivated the PI3K/AKT and Wnt3a/β-catenin signaling pathways. Our findings suggest that sapylin inhibits proliferation and promotes apoptosis in lung cancer cells, thus providing a new theoretical basis for the treatment of lung cancer.     

PCI-24781 can improve in vitro and in vivo developmental capacity of pig somatic cell nuclear transfer embryos

Objective

To examine the effect of PCI-24781 (abexinostat) on the blastocyst formation rate in pig somatic cell nuclear transferred (SCNT) embryos and acetylation levels of the histone H3 lysine 9 and histone H4 lysine 12.

Results

Treatment with 0.5 nM PCI-24781 for 6 h significantly improved the development of cloned embryos, in comparison to the control group (25.3 vs. 10.5 %, P < 0.05). Furthermore, PCI-24781 treatment led to elevated acetylation of H3K9 and H4K12. TUNEL assay and Hoechst 33342 staining revealed that the percentage of apoptotic cells in blastocysts was significantly lower in PCI-24781-treated SCNT embryos than in untreated embryos. Also, PCI-24781-treated embryos were transferred into three surrogate sows, one of whom became pregnant and two fetuses developed.

Conclusion

PCI-24781 improves nuclear reprogramming and the developmental potential of pig SCNT embryos.

     

Roles of sphingosine-1-phosphate (S1P) receptors in malignant behavior of glioma cells. Differential effects of S1P2 on cell migration and invasiveness

Sphingosine-1-phosphate (S1P) is a bioactive lipid that signals through a family of five G-protein-coupled receptors, termed S1P(1-5). S1P stimulates growth and invasiveness of glioma cells, and high expression levels of the enzyme that forms S1P, sphingosine kinase-1, correlate with short survival of glioma patients. In this study we examined the mechanism of S1P stimulation of glioma cell proliferation and invasion by either overexpressing or knocking down, by RNA interference, S1P receptor expression in glioma cell lines. S1P(1), S1P(2) and S1P(3) all contribute positively to S1P-stimulated glioma cell proliferation, with S1P(1) being the major contributor. Stimulation of glioma cell proliferation by these receptors correlated with activation of ERK MAP kinase. S1P(5) blocks glioma cell proliferation, and inhibits ERK activation. S1P(1) and S1P(3) enhance glioma cell migration and invasion. S1P(2) inhibits migration through Rho activation, Rho kinase signaling and stress fiber formation, but unexpectedly, enhances glioma cell invasiveness by stimulating cell adhesion. S1P(2) also potently enhances expression of the matricellular protein CCN1/Cyr61, which has been implicated in tumor cell adhesion, and invasion as well as tumor angiogenesis. A neutralizing antibody to CCN1 blocked S1P(2)-stimulated glioma invasion. Thus, while S1P(2) decreases glioma cell motility, it may enhance invasion through induction of proteins that modulate glioma cell interaction with the extracellular matrix.     

Vitamin K1 enhances sorafenib-induced growth inhibition and apoptosis of human malignant glioma cells by blocking the Raf/MEK/ERK pathway

ABSTRACT: BACKGROUND: The combined effects of anticancer drugs with nutritional factors against tumor cells have been reported previously. This study characterized the efficacy and possible mechanisms of the combination of sorafenib and vitamin K1 (VK1) on glioma cell lines. METHODS: We examined the effects of sorafenib, VK1 or their combination on the proliferation and apoptosis of human malignant glioma cell lines (BT325 and U251) by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, flow cytometry and 4',6-diamidino-2-phenylindole (DAPI) assay. The signaling pathway changes were detected by western blotting. RESULTS: Sorafenib, as a single agent, showed antitumor activity in a dose-dependent manner in glioma cells, but the effects were more pronounced when used in combination with VK1 treatment. Sorafenib in combination with VK1 treatment produced marked potentiation of growth inhibition and apoptosis, and reduced expression of phospho-mitogen-activated protein kinase kinase (MEK) and phospho-extracellular signal-regulated kinase (ERK). Furthermore, the expression levels of antiapoptotic proteins Bcl-2 and Mcl-1 were significantly reduced. CONCLUSIONS: Our findings indicated that VK1 enhanced the cytotoxicity effect of sorafenib through inhibiting the Raf/MEK/ERK signaling pathway in glioma cells, and suggested that sorafenib in combination with VK1 maybe a new therapeutic option for patients with gliomas.     

MicroRNA-144-3p inhibits proliferation and induces apoptosis of human salivary adenoid carcinoma cells via targeting of mTOR

Objectives

To investigate the biological functions of microRNA-144-3p with respect to proliferation and apoptosis of human salivary adenoid carcinoma cell lines via mTOR.

Results

After transfection of microRNA-144-3p agomir, cell viability assays confirmed that the salivary adenoid carcinoma cell (SACC) proliferation was inhibited and apoptosis was induced. Dual luciferase reporter assay validated that the mammalian target of rapamycin (mTOR) was a direct target of miR-144-3p. Western blot, immunofluorescent analysis and a xenograft mouse model of adenoid cystic carcinoma indicated that miR-144-3p was a tumor suppressor and repressed mTOR expression and signaling in SACCs.

Conclusions

MicroRNA-144-3p inhibits proliferation and induces apoptosis of human salivary adenoid carcinoma cells by downregulating mTOR expression in vitro and in vivo.

     

ROS accumulation contributes to abamectin‐induced apoptosis and autophagy via the inactivation of PI3K/AKT/mTOR pathway in TM3 Leydig cells

Abamectin (ABA) as one of the worldwide used compounds in agriculture has raised safety concerns on nontarget organism toxicity. However, the study of male reproductive system damage caused by ABA remains unclear. Our aim is to investigate the effect of ABA‐induced cytotoxicity in TM3 Leydig cells and their underlying mechanisms. ABA inhibits TM3 cell viability and proliferation via cell cycle arrested in the G0/G1 phase. In addition, ABA‐induced mitochondrial depolarization leads to an imbalance in Bcl‐2 family expression, causing caspase‐dependent apoptosis in TM3 cells. The increased ratio of cells expression LC3 protein and LC3‐II to LC3‐I indicated the activation of autophagy potentially. Further experiments revealed ABA treatment reduced phosphatidylinositol 3‐kinase (PI3K), protein kinase B (AKT) phosphorylation, and mammalian target of rapamycin (mTOR) phosphorylation. Pretreatment with a PI3K/AKT inhibitor, LY294002, mimicked the ABA‐mediated effects on cytotoxicity. Pretreatment with a PI3K/AKT agonist, insulin‐like growth factor‐1, reversed the effects of ABA. ABA caused the accumulation of intracellular reactive oxygen species (ROS) by increased intensity of the ROS indicator. However, N‐acetylcysteine as ROS scavengers inhibited ABA‐induced apoptosis and autophagy and reversed these ABA‐mediated effects on PI3K/AKT/mTOR pathway. On the basis of the above results, it is suggested that ABA exposure induces apoptosis and autophagy in TM3 cells by ROS accumulation to mediate PI3K/AKT/mTOR signaling pathway suppression.     

Activation of AMP-activated protein kinase by temozolomide contributes to apoptosis in glioblastoma cells via p53 activation and mTORC1 inhibition

Methylating drugs such as temozolomide (TMZ) are widely used in the treatment of brain tumors including malignant glioblastoma. The mechanism of TMZ-induced glioblastoma cell death and apoptosis, however, is not fully understood. Here, we tested the potential involvement of AMP-activated protein kinase (AMPK) in this process. We found that methylating agents TMZ and N-methyl-N'-nitro-N-nitrosoguanidine induce AMPK activation in primary cultured human glioblastoma and glioblastoma cell lines. TMZ-induced O(6)-methylguanine production is involved in AMPK activation. O(6)-benzylguanine, an O(6)-methylguanine-DNA methyltransferase inhibitor, enhances TMZ-induced O(6)-methylguanine production, leading to enhanced reactive oxygen species production, which serves as an upstream signal for AMPK activation. Activation of AMPK is involved in TMZ-induced glioblastoma cell death and apoptosis. AMPK inhibitor (Compound C) or AMPKα siRNA knockdown inhibits TMZ-induced glioblastoma cell death and apoptosis, whereas AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside enhances it. In further studies, we found that activation of AMPK is involved in TMZ-induced p53 activation and subsequent p21, Noxa, and Bax up-regulation. Activation of AMPK by TMZ also inhibits mTOR complex 1 (mTORC1) signaling and promotes anti-apoptosis protein Bcl-2 down-regulation, which together mediate TMZ-induced pro-cell apoptosis effects. Our study suggests that activation of AMPK by TMZ contributes to glioblastoma cell apoptosis, probably by promoting p53 activation and inhibiting mTORC1 signaling.     

LncRNA MYU对胶质瘤细胞周期分布、增殖、转移和凋亡以及PI3K/Akt信号通路的影响

摘要 目的：探讨长链非编码RNA（LncRNA）MYU对胶质瘤细胞周期分布、细胞增殖、迁移、侵袭和凋亡的影响,并初步探讨其作用机制。方法：实时荧光定量PCR（RT-qPCR）检测人脑正常胶质细胞HEB和胶质瘤细胞（U-251MG、A172、SHG139）中LncRNA MYU的表达情况。选取SHG139细胞,分为正常对照（NC）组、si-con组、si-LncRNA MYU组进行转染实验,行RT-qPCR检测转染效果。分别采用流式细胞术、细胞计数试剂盒（CCK-8）、Transwell实验检测沉默LncRNA MYU对SHG139细胞周期分布和凋亡、细胞增殖、细胞迁移和侵袭的影响。蛋白免疫印迹（Western blot）法检测基质金属蛋白酶2（MMP-2）、MMP-9、裂解的半胱氨酸天冬氨酸蛋白酶3（Cleaved caspase-3）、Cleaved caspase-9以及磷脂酰肌醇-3-羟激酶/蛋白激酶B（PI3K/Akt）信号通路相关蛋白表达情况。结果：LncRNA MYU在胶质瘤细胞株中比人脑正常胶质细胞中的表达水平显著升高（P＜0.05）,因此选择表达量最高的SHG139细胞进行转染实验。沉默LncRNA MYU能够显著诱导SHG139细胞G0-G1期阻滞、抑制细胞增殖、迁移和侵袭并诱导细胞凋亡（P＜0.05）。沉默LncRNA MYU可显著抑制MMP-2、MMP-9、p-PI3K和p-AKT表达并促进Cleaved caspase-3、Cleaved caspase-9表达（P＜0.05）。结论：沉默LncRNA MYU可诱导胶质瘤细胞G0-G1期阻滞,抑制细胞增殖、迁移和侵袭,促进细胞凋亡,其机制可能与抑制PI3K/AKT信号通路有关。     

LRRC4 inhibits the proliferation of human glioma cells by modulating the expression of STMN1 and microtubule polymerization

LRRC4 is a tumor suppressor of glioma, and it is epigenetically inactivated commonly in glioma. Our previous study has shown that induction of LRRC4 expression inhibits the proliferation of glioma cells. However, little is known about the mechanisms underlying the action of LRRC4 in glioma cells. We employed two-dimensional fluorescence differential gel electrophoresis (2-D DIGE) and MALDI -TOF/TOF-MS/MS to identify 11 differentially expressed proteins, including the significantly down-regulated STMN1 expression in the LRRC4-expressing U251 glioma cells. The levels of STMN1 expression appeared to be positively associated with the pathogenic degrees of human glioma. Furthermore, induction of LRRC4 over-expression inhibited the STMN1 expression and U251 cell proliferation in vitro, and the glioma growth in vivo. In addition, induction of LRRC4 or knockdown of STMN1 expression induced cell cycle arrest in U251 cells, which was associated with modulating the p21, cyclin D1, and cyclin B expression, and the ERK phosphorylation, and inhibiting the CDK5 and cdc2 kinase activities, but increasing the microtubulin polymerization in U251 cells. LRRC4, at least partially by down-regulating the STMN1expression, acts as a major glioma suppressor, induces cell cycle arrest and modulates the dynamic process of microtubulin, leading to the inhibition of glioma cell proliferation and growth. Potentially, modulation of LRRC4 or STMN1 expression may be useful for design of new therapies for the intervention of glioma.     

Enhancer of zeste homolog 2 promotes the proliferation and invasion of epithelial ovarian cancer cells

Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 (PRC2) that includes noncatalytic subunits suppressor of zeste 12 (SUZ12) and embryonic ectoderm development (EED). When present in PRC2, EZH2 catalyzes trimethylation on lysine 27 residue of histone H3 (H3K27Me3), resulting in epigenetic silencing of gene expression. Here, we investigated the expression and function of EZH2 in epithelial ovarian cancer (EOC). When compared with primary human ovarian surface epithelial (pHOSE) cells, EZH2, SUZ12, and EED were expressed at higher levels in all 8 human EOC cell lines tested. Consistently, H3K27Me3 was also overexpressed in human EOC cell lines compared with pHOSE cells. EZH2 was significantly overexpressed in primary human EOCs (n = 134) when compared with normal ovarian surface epithelium (n = 46; P < 0.001). EZH2 expression positively correlated with expression of Ki67 (P < 0.001; a marker of cell proliferation) and tumor grade (P = 0.034) but not tumor stage (P = 0.908) in EOC. There was no correlation of EZH2 expression with overall (P = 0.3) or disease-free survival (P = 0.2) in high-grade serous histotype EOC patients (n = 98). Knockdown of EZH2 expression reduced the level of H3K27Me3 and suppressed the growth of human EOC cells both in vitro and in vivo in xenograft models. EZH2 knockdown induced apoptosis of human EOC cells. Finally, we showed that EZH2 knockdown suppressed the invasion of human EOC cells. Together, these data demonstrate that EZH2 is frequently overexpressed in human EOC cells and its overexpression promotes the proliferation and invasion of human EOC cells, suggesting that EZH2 is a potential target for developing EOC therapeutics.