Protein Expression Levels Of c-Myc Research Articles

Arenobufagin inhibits lung metastasis of colorectal cancer by targeting c-MYC/Nrf2 axis

Colorectal cancer (CRC) is one of the commonest cancers worldwide. Metastasis is the most common cause of death in patients with CRC. Arenobufagin is an active component of bufadienolides, extracted from toad skin and parotid venom. Arenobufagin reportedly inhibits epithelial-to-mesenchymal transition (EMT) and metastasis in various cancers. However, the mechanism through which arenobufagin inhibits CRC metastasis remains unclear. This study aimed to elucidate the molecular mechanisms by which arenobufagin inhibits CRC metastasis. Wound-healing and transwell assays were used to assess the migration and invasion of CRC cells. The expression of nuclear factor erythroid-2-related factor 2 (Nrf2) in the CRC tissues was assessed using immunohistochemistry. The protein expression levels of c-MYC and Nrf2 were detected by immunoblotting. A mouse model of lung metastasis was used to study the effects of arenobufagin on CRC lung metastasis in vivo. Arenobufagin observably inhibited the migration and invasion of CRC cells by downregulating c-MYC and inactivating the Nrf2 signaling pathway. Pretreatment with the Nrf2 inhibitor brusatol markedly enhanced arenobufagin-mediated inhibition of migration and invasion, whereas pretreatment with the Nrf2 agonist tert‑butylhydroquinone significantly attenuated arenobufagin-mediated inhibition of migration and invasion of CRC cells. Furthermore, Nrf2 knockdown with short hairpin RNA enhanced the arenobufagin-induced inhibition of the migration and invasion of CRC cells. Importantly, c-MYC acts as an upstream modulator of Nrf2 in CRC cells. c-MYC knockdown markedly enhanced arenobufagin-mediated inhibition of the Nrf2 signaling pathway, cell migration, and invasion. Arenobufagin inhibited CRC lung metastasis in vivo. Together, these findings provide evidence that interruption of the c-MYC/Nrf2 signaling pathway is crucial for arenobufagin-inhibited cell metastasis in CRC. Collectively, our findings show that arenobufagin could be used as a potential anticancer agent against CRC metastasis. The arenobufagin-targeted c-MYC/Nrf2 signaling pathway may be a novel chemotherapeutic strategy for treating CRC.

Buffalo Milk Whey Activates Necroptosis and Apoptosis in a Xenograft Model of Colorectal Cancer.

Recent pharmacological research on milk whey, a byproduct of the dairy industry, has identified several therapeutic properties that could be exploited in modern medicine. In the present study, we investigated the anticancer effects of whey from Mediterranean buffalo (Bubalus bubalis) milk. The antitumour effect of delactosed milk whey (DMW) was evaluated using the HCT116 xenograft mouse model of colorectal cancer (CRC). There were no discernible differences in tumour growth between treated and untreated groups. Nevertheless, haematoxylin and eosin staining of the xenograft tissues showed clearer signs of different cell death in DMW-treated mice compared to vehicle-treated mice. Detailed biochemical and molecular biological analyses revealed that DMW was able to downregulate the protein expression levels of c-myc, phospho-Histone H3 (ser 10) and p-ERK. Moreover, DMW also activated RIPK1, RIPK3, and MLKL axis in tumour tissues from xenograft mice, thus, suggesting a necroptotic effect. The necroptotic pathway was accompanied by activation of the apoptotic pathway as revealed by increased expression of both cleaved caspase-3 and PARP-1. At the molecular level, DMW-induced cell death was also associated with (i) upregulation of SIRT3, SIRT6, and PPAR-γ and (ii) downregulation of LDHA and PPAR-α. Overall, our results unveil the potential of whey as a source of biomolecules of food origin in the clinical setting of novel strategies for the treatment of CRC.

Ligustilide from Radix Angelica Sinensis prevents the migration of vascular smooth muscle A7r5 cells based on network pharmacology and experimental verification

ObjectiveLigustilide (Lig), a major component of Radix Angelica Sinensis, exhibits a protective effect on many cardiovascular diseases. Our previous study showed that Lig inhibited the proliferation of vascular smooth muscle cells. In this study, we aimed to investigate the effect of Lig on the migration of vascular smooth muscle A7r5 cells induced by Angiotensin II (Ang II) and explore the mechanism underlying based on network pharmacology. MethodsIn this study, scratch-wound healing assay and transwell migration assay were used to assess the migration activity of Lig on Ang II-induced vascular smooth muscle A7r5 cells, and network pharmacology method was used to predict the possible target molecules. Western blot analysis, Gelatin zymography assay, and small interfering RNA (siRNA) were used to determine the underlying mechanism of Lig on the migration in Ang II-induced A7r5 cells. ResultsWe found that Lig could prevented the migration induced by Ang II in A7r5 cells, which could be associated with c-Myc and MMPs from the results of network pharmacology. Our results revealed that Lig could significantly reduce the increase in the protein expression levels of c-Myc and MMP-2 in Ang II-induced A7r5 cells, but not affect the protein expression levels of MMP-9. Our data further showed that c-Myc siRNA, just as Lig, could obviously inhibit the cell migration accompanied by decreased MMP-2 expression. ConclusionThese findings suggested the anti-migratory effect of Lig could be associated with the c-Myc/MMP-2 pathway, and Lig administration had a promising potential for preventing cardiovascular diseases.

Angiotensin II promotes primary tumor growth and metastasis formation of murine TNBC 4T1 cells through the fibroblasts around cancer cells

Angiotensin II (Ang II) reportedly facilitates primary tumor growth and distal hematogenous metastasis formation in various murine intravenous metastasis models. However, it is unclear whether Ang II accelerates the initial processes of metastasis formation that begins in primary tumors surrounded by tumor microenvironment. We examined the effects of Ang II on primary tumors and lung metastasis lesions using a murine spontaneous metastasis model, in which triple negative breast cancer 4T1 cells constitutively expressing luciferase (4T1-Luc cells) were injected into the mammary fat pad of BALB/c mice. Subcutaneous injection of Ang II significantly accelerated primary tumor growth and lung metastasis formation. Ang II increased the protein expression levels of c-Myc, cyclin D1, fibronectin, vimentin, αSMA and Snail, and the treatment with the Ang II type 1 receptor blocker valsartan significantly suppressed the Ang II-induced increases of fibronectin and vimentin. Valsartan also significantly reduced lung metastatic lesions. However, Ang II did not have significant effects on 4T1-Luc cells including the proliferation, migration, invasion, or the expressions of proteins related to cell proliferation and epithelial-to-mesenchymal transition. In contrast, when 4T1-Luc cells were co-cultured with dermal fibroblasts, Ang II significantly accelerated cell migration and increased the expressions of fibronectin, vimentin, αSMA and Snail in 4T1-Luc cells. And moreover, Ang II significantly increased the mRNA expression of IL-6 in fibroblasts co-cultured with 4T1-Luc cells. These results suggested that Ang II accelerates surrounding fibroblasts by soluble factors such as IL-6 to promote epithelial-to-mesenchymal transition, which result in the initiation of cancer metastasis.

Macleayins A From Macleaya Promotes Cell Apoptosis Through Wnt/β-Catenin Signaling Pathway and Inhibits Proliferation, Migration, and Invasion in Cervical Cancer HeLa Cells.

Macleayins A (MA), a novel compound, was isolated from Macleaya cordata (Willd.) R. Br. and Macleaya microcarpa (Maxim.) Fedde. The plant species are the member of Papaveraceae family and have been used traditionally for diverse therapeutic purposes. According to the reported studies, the chemical constituents, as well as crude extracts of these plants, could attenuate the proliferation of several cancer cell lines, such as HL-60, A549, HepG2, and MCF-7. The current study aimed to investigate the anticervical cancer activity of MA and its related molecular mechanism. Isolation of MA was carried out using various column chromatographic methods, and its structure was elucidated with 1H NMR. The cytotoxicity of MA was determined against HeLa cell lines via CCK-8 assay. The cell proliferation, apoptosis, cell cycle, migration, and invasion were measured by EdU labeling, Annexin-V APC/7-AAD double staining, PI staining, and transwell assay, respectively. The protein expression levels of c-Myc, β-catenin, cyclin D1, and MMP-7 in the cells were evaluated by western blotting. The Wnt/β-catenin signaling cascade activation was verified using the Dual-Glo® Luciferase assay. We found that MA inhibited the growth of HeLa cells at 72 h (IC50 = 26.88 µM) via inducing apoptotic process, reduced the proliferation rate by 29.89%, and decreased the cells migration and invasion as compared to the untreated group. It arrested the cell cycle at the G1 phase and its treatment inhibited the expression of related proteins c-Myc, β-catenin, cyclin D1, and MMP-7 in the Wnt/β-catenin signaling cascade. Further, the Wnt/β-catenin signaling cascade activation in MA-treated HeLa cells was attenuated in a dose-dependent manner. These findings demonstrate the anticancer effects of MA on a mechanistic level, thus providing a basis for MA to become a potential candidate drug for resistance of cervical carcinoma.

Salidroside overcomes dexamethasone resistance in T-acute lymphoblastic leukemia cells.

The aim of the present study was to analyze whether the use of salidroside (SAL) could overcome dexamethasone (DEX) resistance in T-acute lymphocytic leukemia cells. The human T-ALL DEX-resistant cell line, CEM-C1 and the DEX-sensitive cell line, CEM-C7 were used in the current study. The proliferation inhibition rates in these cells, treated with SAL and DEX alone, and in combination were detected using a Cell Counting Kit-8 assay, while the morphological changes of the cells were observed using an inverted microscope. Reverse transcription-quantitative PCR was used to detect the mRNA expression levels of the c-Myc and LC3 genes, while flow cytometry was used to detect the cell cycle distribution and the rate of apoptosis. In addition, western blot analysis was used to detect the protein expression levels of c-Myc, BCL-2, Bax, cleaved PARP and LC3. and acridine orange staining was used to detect the changes in acidic autophagy vesicles. It was found that SAL could effectively inhibit cell proliferation and induce apoptosis in the CEM-C1 and CEM-C7 cells. In addition, SAL promoted the induction of autophagy. The protein expression levels of c-Myc in the CEM-C1 cells were significantly higher compared with that in the CEM-C7 cells. SAL downregulated the mRNA expression levels of the c-Myc gene and protein in a dose-dependent manner. This suggested that SAL could inhibit the proliferation of the CEM-C1 and CEM-C7 cells, induce apoptosis and autophagy and overcome DEX resistance in the CEM-C1 cells. The mechanism may be associated with the downregulation of c-Myc.

Long non-coding RNA PLAC2 suppresses the survival of gastric cancer cells through down-regulating C-Myc.

The aim of this study was to explore the effects of long non-coding ribonucleic acid (lncRNA) placenta-specific protein 2 (PLAC2) on the biological behaviors of gastric cancer (GC) cells by regulating the expression of c-Myc gene and its mechanism. The expression of PLAC2 in GC tissues and different GC cell lines was detected via quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). The effects of PLAC2 on apoptosis and cycle, migration, and invasion of GC cells were detected using flow cytometry, wound healing assay, and transwell assay, respectively. After interference in PLAC2 expression, the changes in c-Myc expression were determined through qRT-PCR and Western blotting. The expression level of PLAC2 was downregulated in 38 out of 45 cases of GC tissues compared with that in normal gastric tissues, and it also declined in GC cells. The results of flow cytometry showed that after overexpression of PLAC2, the cell cycle was arrested in the G1/G0 phase, and the apoptosis rate was increased. The results of wound healing assay and transwell assay revealed that both migration and invasion of GC cells were inhibited. After overexpression of PLAC2, the mRNA and protein expression levels of c-Myc declined. LncRNA PLAC2 affects the biological behaviors of GC cells by regulating the expression of c-Myc gene.

Targeting both BET and CBP/EP300 proteins with the novel dual inhibitors NEO2734 and NEO1132 leads to anti-tumor activity in multiple myeloma.

Two promising epigenetic therapeutic targets have emerged for the treatment of hematologic malignancies, BET and CBP/EP300 proteins. Several studies have shown that targeting these individual classes of proteins has anti-tumor activity in multiple myeloma (MM), as well as other cancers. Here, we present the first data exploring the anti-tumor activity of two novel dual inhibitors, NEO2734 and NEO1132, of both BET and CBP/EP300 proteins in MM. Sixteen MM cell lines (MMCLs) were treated with the dual inhibitors NEO2734 and NEO1132, the single BET inhibitors JQ1, OTX015, IBET-762, and IBET-151, and a single CBP/EP300 inhibitor CPI-637. The dual inhibitor NEO2734 showed strong anti-tumor activity and was consistently highly active against all MMCLs, being as potent as JQ1 and more so than other single inhibitors. NEO2734 and NEO11132 induced a significant G1 cell cycle arrest and decreased c-MYC and IRF4 protein levels in MMCLs compared to the other single inhibitors. Sensitivity to the dual inhibitors was not dependent on a specific MM molecular subgroup but correlated with c-MYC protein expression levels. The dual inhibition of BET and CBP/EP300 has potential therapeutic benefits for patients with MM.

Synergistic Carcinogenesis of HPV18 and MNNG in Het-1A Cells through p62-KEAP1-NRF2 and PI3K/AKT/mTOR Pathway.

N-methyl-N´-nitro-N-nitrosoguanidine is a clear carcinogen, increasing evidence that indicates an etiological role of human papillomavirus in esophageal carcinoma. Studies have reported the synergistic effect on environmental carcinogens and viruses in recent years. On the basis of establishing the malignant transformation model of Het-1A cells induced by synergistic of HPV18 and MNNG, this study was to explore the synergistic carcinogenesis of MNNG and HPV. Our research indicated that HPV&MNNG led to a significant increase in the protein-expression levels of c-Myc, cyclinD1, BCL-2, BAX, E-cadherin, N-cadherin, mTOR, LC3II, and p62, with concomitant decreases in p21 and LC3I. HPV18 and MNNG induced accumulation of p62 and its interaction with KEAP1, which promoted NRF2 nuclear translocation. p62 loss prevents growth and increases autophagy of malignant cells by activating KEAP1/NRF2-dependent antioxidative response. In addition, PI3K and p-AKT were stimulated by HPV&MNNG, and PI3K/AKT/mTOR is positively associated with cell proliferation, migration, invasion, and autophagy during malignant transformation. Taken together, MNNG&HPV regulates autophagy and further accelerates cell appreciation by activating the p62/KEAP1/NRF2 and PI3K/AKT/mTOR pathway. MNNG&HPV may improve Het-1A cell autophagy to contribute to excessive cell proliferation, reduced apoptosis, and protection from oxidative damage, thus accelerating the process of cell malignant transformation and leading to cancerous cells.

Inhibition of scutellarin on differentiation of colonic cancer stem cells via hedgehog signaling pathway

The objective of this study was to investigate the inhibitory effect of scutellarin on the differentiation of colonic cancer stem cells in vitro and in vivo and to explore its underlying hedgehog signaling-based mechanism. The effect of scutellarin on the growth in vitro of HT-29 cells-derived cancer stem-like cells(HT-29 CSC) was observed with 3 D cell culture. The effect of scutellarin on the transformation of HT-29 CSC cells was assessed by soft agar colony formation assay. Fetal calf serum was used to induce differentiation of stem cells and observe the effect of scutellarin on HT-29 CSC cells differentiation in vitro. The effects of scutellarin on mRNA expressions of Lgr5, c-Myc, CK20 and Nanog in HT-29 CSC cells were determined by quantitative Real-time polymerase chain reaction(qRT-PCR). The effects of scutellarin on protein expressions of c-Myc, Gli1 and Lgr5 in HT-29 CSC cells were examined by Western blot. After subcutaneous implantation of HT-29 CSC cells in nude mice, the effect of scutellarin on the mouse body weight and the growth of HT-29 CSC-derived tumor were explored. qRT-PCR was used for evaluating the effect of scutellarin on mRNA levels of CD133, Lgr5, Gli1, Ptch1, c-Myc, Ki-67, CK20 and Nanog in tumor. Western blot and immunohistochemistry analysis were used to detect the effect of scutellarin on protein expressions of c-Myc, Gli1, Lgr5, CD133 and Ki-67 in tumor. The in vitro experiments showed that scutellarin inhibited the growth, transformation and differentiation of HT-29 CSC cells, significantly down-regulated the mRNA levels of Lgr5, c-Myc, CK20 and Nanog in HT-29 CSC cells as well as the protein expression levels of c-Myc, Gli1 and Lgr5 in HT-29 CSC cells. Additionally, animal experiments showed that scutellarin significantly inhibited the growth of subcutaneous xenografts in nude mice, and down-regulated the mRNA expressions of CD133, Lgr5, Gli1, Ptch1, c-Myc, Ki-67, CK20 and Nanog as well as the protein levels of c-Myc, Gli1, Lgr5, CD133 and Ki-67 of xenografts in nude mice. Taken together, scutellarin could inhibit the differentiation of colo-nic cancer stem cells in vitro and in vivo, potentially by down regulation of hedgehog signaling pathway activity.

Effects of Laminaria Japonica Polysaccharides on the Survival of Non-Small-Cell Lung Cancer A549 Cells

Objective. To investigate the effect of Laminaria japonica polysaccharides (LJP) on the survival of non-small-cell lung cancer (NSCLC) A549 cells and its mechanism. Methods. In vitro: the cells were randomly divided into control group, LJP (5 mg/ml) group, LJP (10 mg/ml) group, and LJP (20 mg/ml) group. After corresponding treatment, the survival rate and the expression of proteins related to proliferation, apoptosis, epithelial-mesenchymal transition (EMT), and signaling pathway were detected by CCK8 assay and Western blot, respectively. In vivo: a xenograft model was established to detect the tumor volume and mass and the expression of the above pathway proteins. Results. Compared with the control group, LJP decreased the survival rate of A549 cells (P<0.05), inhibited the protein expression of Ki67 and PCNA (P<0.05), downregulated the expression of Bcl-2 while upregulated the expression of Bax, cl-caspase-3, and cl-caspase-9 (P<0.05), upregulated the expression of E-cadherin, downregulated the expression of vascular endothelial growth factor (VEGF) and N-cadherin (P<0.05), and downregulated β-catenin, transcription factor-4 (TCF4), and c-Myc protein expression levels (P<0.05). In vivo: LJP decreased the volume and mass of the xenograft tumors and downregulated β-catenin, TCF4, and c-Myc protein expression levels compared with the control group (P<0.05). Conclusion. LJP can inhibit the survival of non-small-cell lung cancer A549 cells in vitro, and its mechanism is related to the inhibition of activation of β-catenin/TCF4 pathway activation.

MiR-760 exerts an antioncogenic effect in esophageal squamous cell carcinoma by negatively driving fat metabolism via targeting c-Myc.

miR-760 is downregulated in various human tumors, and fat metabolism disorder correlates with tumor progression, especially anomalism of key fat metabolic enzymes that are positively modulated by c-Myc. The aim of our study is to elucidate the presumptive molecular mechanisms of miR-760-mediated esophageal squamous cell carcinoma (ESCC) cell function and to assess the therapeutic significance of miR-760 in ESCC patients. Quantitative real-time PCR (RT-qPCR) analysis indicated that miR-760 was significantly downexpressed in ESCC tissues and cell lines. Cell counting kit-8 (CCK-8) assay, colony formation assay, transwell assay, and flow cytometry denoted that induced ectopic overexpression of miR-760 dramatically inhibited ESCC cells proliferation, attenuated migration, and invasion facilitated apoptosis in vitro. Mechanistically, c-Myc predicted using bioinformatics was identified as a potential target gene of miR-760 by luciferase reporter assay. Furthermore, mRNA and protein expression levels of c-Myc and key fat metabolic enzymes were downregulated with miR-760 mimics. The above investigation results, responsible for the antineoplastic properties of miR-760 in ESCC, preliminarily highlighted that the hypothetical signal amongst miR-760, c-Myc, and key fat metabolic enzymes may develop a novel diagnostic marker, therapeutic target, and independent prognostic indicator.

Knockdown of long non-coding RNA PVT1 inhibits the proliferation of Raji cells through cell cycle regulation.

Long non-coding RNA plasmacytoma variant translocation 1 (PVT1) has been reported to be associated with oncogenesis. However, the functional role of PVT1 in Burkitt lymphoma has not yet been addressed. The purpose of the present study was to investigate the effect of PVT1 knockdown by small interfering RNA (siRNA) on the proliferation of Burkitt lymphoma Raji cells and to explore its possible mechanism of action. An effective siRNA targeting PVT1 was screened and the corresponding short hairpin RNA (shRNA) was reconstructed into a lentiviral vector. Cell proliferation and cell cycle distribution were assessed by Cell Counting kit-8 assay and flow cytometry, respectively. Protein expression levels of c-Myc, cyclin-dependent kinase inhibitor1A (CDKN1A, P21) and cyclin E1 (CCNE1) were detected by western blotting. A polymerase chain reaction (PCR) array was used to analyse the expression of genes associated with the cell cycle. PVT1 knockdown markedly suppressed proliferation, and induced cell cycle arrest at the G0/G1 phase in Raji cells. Protein expression levels of c-Myc and CCNE1 were reduced, whereas P21 protein expression was markedly increased following downregulation of PVT1 in Raji cells. The cell cycle PCR array revealed that 54 genes were upregulated and 26 genes were downregulated in Raji cells following PVT1 knockdown. Reverse transcription-quantitative PCR demonstrated that cyclin G2 (CCNG2), CDKN1A, Retinoblastoma-like 2 (RBL2, p130), HUS1 checkpoint homolog, cyclin dependent kinase inhibitor 3 (CDKN3) and cyclin dependent kinase inhibitor 1B (CDKN1B) expression were upregulated, whereas the expression levels of CCNE1, cyclin D1 (CCND1) and cell division cycle 20 (CDC20) were downregulated in Raji cells with PVT1 knockdown. In conclusion, PVT1 knockdown may inhibit the proliferation of Raji cells by arresting cells in G0/G1 phase. Furthermore, inhibition of cell proliferation may be associated with a reduction inc-Myc expression and alterations in the expression levels of cell cycle-associated genes.

NOTCH signaling is activated in and contributes to resistance in enzalutamide-resistant prostate cancer cells

Prostate cancer is the second leading cause of cancer death among men in the United States. The androgen receptor (AR) antagonist enzalutamide is a Food and Drug Administration-approved drug for treatment of patients with late-stage prostate cancer and is currently under clinical study for early-stage prostate cancer treatment. After a short positive response period, tumors will develop drug resistance. In this study using RNA-Seq and bioinformatics analyses, we observed that NOTCH signaling is a deregulated pathway in enzalutamide-resistant cells. NOTCH2 and c-MYC gene expression positively correlated with AR expression in samples from patient with hormone refractory disease in which AR expression levels correspond to those typically observed in enzalutamide resistance. Cleaved NOTCH1, HES1 (Hes family BHLH transcription factor 1), and c-MYC protein expression levels are elevated in two enzalutamide-resistant cell lines, MR49F and C4-2R, indicating NOTCH signaling activation. Moreover, inhibition of the overexpressed ADAM metallopeptidase domain 10 (ADAM10) in the resistant cells induces an exclusive reduction in cleaved NOTCH1 expression. Furthermore, exposure of enzalutamide-resistant cells to both PF-03084014 and enzalutamide increased cell death, decreased colony formation ability, and resensitized cells to enzalutamide. Knockdown of NOTCH1 in C4-2R increased enzalutamide sensitivity by decreasing cell proliferation and increasing cleaved PARP expression. In a 22RV1 xenograft model, PF-03084014 and enzalutamide decreased tumor growth through reducing cell proliferation and increasing apoptosis. These results indicate that NOTCH1 signaling may contribute to enzalutamide resistance in prostate cancer, and inhibition of NOTCH signaling can resensitize resistant cells to enzalutamide.

MicroRNA-940 Targets INPP4A or GSK3β and Activates the Wnt/β-Catenin Pathway to Regulate the Malignant Behavior of Bladder Cancer Cells.

In this report, we aimed to explore the role and regulatory mechanism of microRNA-940 (miR-940) in bladder cancer development. The expressions of miR-940 in bladder cancer tissues and cells were measured. miR-940 mimics, miR-940 inhibitor small interference RNA against INPP4A (si-INPP4A), and GSK3β (si-GSK3β) and their corresponding controls were then transfected into cells. We investigated the effects of miR-940, INPP4A, or GSK3β on cell proliferation, migration, invasion, and apoptosis. Additionally, target prediction and luciferase reporter assays were performed to investigate the targets of miR-940. The regulatory relationship between miR-940 and the Wnt/β-catenin pathway was also investigated. miR-940 was upregulated in bladder cancer tissues and cells. Overexpression of miR-940 significantly increased bladder cancer cell proliferation, promoted migration and invasion, and inhibited cell apoptosis. INPP4A and GSK3β were the direct targets of miR-940, and knockdown of INPP4A or GSK3β significantly increased cancer cell proliferation, migration, and invasion and inhibited cell apoptosis. After miR-940 overexpression, the protein expression levels of c-Myc, cyclin D1, and β-catenin were significantly increased, and the expression levels of p27 and p-β-catenin were markedly decreased. The opposite effects were obtained after suppression of miR-940. XAV939, a tankyrase 1 inhibitor that could inhibit Wnt/β-catenin signaling, significantly reversed the effects of miR-940 overexpression on cell migration and invasion. Our results indicate that overexpression of miR-940 may promote bladder cancer cell proliferation, migration, and invasion and inhibit cell apoptosis via targeting INPP4A or GSK3β and activating the Wnt/β-catenin pathway. Our findings imply the key roles of suppressing miRNA-940 in the therapy of bladder cancer.

Abstract 3349: A regulatory circuit HP1 gamma/miR-451a/c-Myc promotes prostate cancer progression

Abstract Heterochromatin protein 1 gamma (HP1 gamma), as a reader of the histone codes, is involved in the determination of higher order of chromatin structure. It has also been implicated in carcinogenesis of various cancer types. However, the role of HP1 gamma in prostate cancer (PCa) progression and the underlying molecular mechanisms remain largely unknown. Using the public microarray data from GEO and TCGA database, we found that HP1 gamma was upregulated in PCa, comparing with benign prostate tissues. PCa patients with higher level of HP1 gamma had shorter disease-free survival, indicating that HP1 gamma level can serve as prognostic marker in PCa patients (p = 0.016). In addition, depletion of HP1 gamma by shRNA in PCa cells not only repressed proliferation and induced apoptosis, but also impaired tumorigenicity in vitro and in vivo. We also found that c-Myc was capable of upregulating HP1 gamma by directly binding to the E-box element in the first exon of HP1 gamma gene, and the upregulated HP1 gamma, in turn, repressed the expression of miR-451a by enhancing H3K9 methylation at the promoter region of miR-451a. Furthermore, reduction of miR-451a significantly reversed HP1 gamma loss-induced PCa cell apoptosis, whereas miR-451a overexpression repressed cell survival by targeting and downregulating c-Myc. The association among mRNA and protein expression levels of c-Myc, HP1 gamma and miR-451a were further confirmed in human clinical samples using Spearman correlation analysis. Therefore, we propose that an HP1 gamma/miR-451a/c-Myc regulatory circuit exists in PCa cells and this circuit plays a crucial role in PCa progression. Citation Format: Ruimin Huang, Cunjie Chang, Jiakuan Liu, Xiaojing Huang, Lan Shen, Dianzheng Zhang, Jun Yan. A regulatory circuit HP1 gamma/miR-451a/c-Myc promotes prostate cancer progression [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 3349. doi:10.1158/1538-7445.AM2017-3349

Abstract 2022: Twist1 regulates UVB-induced epidermal cell proliferation in non-melanoma skin cancer

Abstract Nonmelanoma skin cancer (NMSC) is the most common cancer and consists of basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). SCC metastases account for up to 20% of all deaths from skin cancer. The major risk factor for the development of NMSC is solar ultraviolet radiation (UV). UVA and UVB both function as initiators and promoters in carcinogenesis and possess immunosuppressive activity. Given the prevalence of this disease, there remains a pressing need for the development of novel prevention and treatment strategies for NMSC. In a recent study (Srivastava et al. 2015. Mol. Carcinog. doi:10.1002/mc.22335), we evaluated the effect of Twist1, a transcription factor that is known to play a role in tumor progression, during epithelial carcinogenesis. Using the two-stage chemical carcinogenesis model, we identified that BK5.Cre x Twist1flox/flox mice, which have a keratinocyte-specific Twist1 knockout (Twist1 KO), developed significantly reduced numbers of papillomas (70% reduction) and SCCs (75% reduction) as well as delayed tumor latency compared to wild-type (WT) mice. Twist1 KO mice also exhibited significantly reduced epidermal proliferation in response to chemical promotion that correlated with reduced levels of cell cycle regulators and increased levels of p53 and p21. In further studies, we have begun analyzing the effects of UVB irradiation in Twist1 KO mice. Specifically, Twist1 KO mice exhibited heightened sensitivity to UVB-induced apoptosis and decreased epidermal proliferation as compared to similarly exposed WT mice. In addition, the accumulation of mast cells in the skin was significantly decreased in Twist1 KO mice as compared to WT mice. Further preliminary experiments showed that the protein expression levels of c-Myc, phospho-p65 (S536) and phospho-p50 (S337) decreased following UVB irradiation whereas levels of the cell cycle inhibitors p21 and p27 exhibited an increased expression in Twist1 KO mice as compared to WT mice. Further experiments have suggested that Twist1 may play a pivotal role in the proliferation/migration of bulge region keratinocyte stem cells (KSCs) following exposure to UVB. These and other results from ongoing experiments will be presented. Collectively, these findings suggest that Twist1 may have a novel role in UVB-induced epithelial carcinogenesis by regulating proliferation and migration of keratinocytes and KSCs. Research supported by CPRIT RP150346, CPRIT RP140108, and NIH T32 ES007247. Citation Format: Jaya Srivastava, Okkyung Rho, John DiGiovanni. Twist1 regulates UVB-induced epidermal cell proliferation in non-melanoma skin cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2022.

Genome-wide transcriptome analysis reveals inactivation of Wnt/β-catenin by 3,3'-diindolylmethane inhibiting proliferation of colon cancer cells.

Multiple genetic and signaling pathway alterations underlie the development of colon cancer. We utilized genome-wide transcriptome analysis to identify important gene expression patterns following treatment with 3,3'-diindolylmethane (DIM), a natural compound derived from cruciferous vegetables, on colon cancer cells. Statistical analyses of gene expression data from DIM treated cells revealed that 692genes were significantly upregulated, while 731genes were down-regulated. Putative gene networks showed that several oncogenes (β-catenin, Myc and FOS) were significantly suppressed by DIM treatment. Using clinical data from colon cancer patients, activation of β-catenin was found to be significantly associated with patient prognosis by Kaplan-Meir plot analysis. We validated the mRNA and protein expression levels of c-Myc, β-catenin, and cyclin D1, all of which were significantly suppressed after DIM treatment in DLD-1 and HCT116 cells. System level characterization of our findings suggests for the first time that β-catenin and c-Myc, which are major genes involved in colon carcinogenesis, were significantly downregulated by DIM treatment in colon cancer cells. Therefore, targeting Wnt/β-catenin signaling by DIM may be an attractive strategy for the prevention and treatment of colon cancer.

Abstract 510: Inhibition of IRE1α-driven pro-survival pathways is a promising therapeutic application in acute myeloid leukemia

Abstract Survival of cancer cells rely on the unfolded protein response (UPR) to resist stress triggered by the accumulation of misfolded proteins within the endoplasmic reticulum (ER). IRE1α-XBP1 pathway, one of the most important branches of the UPR, is activated in different types of cancer. Upon IRE1α activation, the spliced form of XBP1 (XBP1s) is generated and acts as a transcription factor for many pro-survival genes to prevent cellular death and relieve cellular stress. Blockade of the IRE1α-XBP1 pathway can be considered as a potential anti-cancer strategy. Acute myeloid leukemia (AML) is the most common acute leukemia in adults. We find that XBP1 and XBP1s are up-regulated in AML cell lines and samples from patients. IRE1α RNase inhibitors including STF-083010, HNA and MKC-204 blocked XBP1 mRNA splicing and exhibited modest cytotoxicity in liquid culture against AML cell lines (HNA, mean IC50, 31 µM, n=8) and AML samples from patients (HNA, mean IC50, 35 µM, n=18). Notably, the IRE1α inhibitor HNA caused a significant inhibition (mean IC50, 6 µM, n=6) of clonogenic growth in soft agar of AML cells from patients of different leukemic subtypes. In contrast, HNA had very little toxicity against normal human marrow committed myeloid colony forming cells (mean IC50, 123 µM, n=4). IRE1α inhibition in AML cells induced caspase-dependent apoptosis and G1 cell cycle arrest which was associated with regulation of Bcl-2 family proteins, G1 phase controlling proteins (p21cip1, p27kip1 and Cyclin D1) and Chaperone proteins (CHOP, HERPUD1, DNAJC3, DNAJB9 and EMDM). Absence of Xbp1 (Cre-induced deletion of floxed XBP1) resulted in the myeloid cells becoming resistant to IRE1α inhibitors. Combination of HNA with either bortezomib or As2O3 synergistically inhibited growth of NB4 acute promyelocytic leukemia cells associated with enhanced levels of p-JNK and reduced expression of p-PI3K and p-MAPK. Also, in a dose- and time- dependent manner, inhibition of IRE1α RNase activity increased expression of many miRNAs (especially miR-17, -21, -34a, -96, -125b and -150) in AML cells. Furthermore, HNA treatment inhibited mRNA levels of several miR-34a targeted genes (c-Myc, Cyclin D1 and CDK4). The inhibition of miR-34a conferred cellular resistance to HNA, as measured by significant increase of cell viability. Addition of a miR-34a antagonist restored protein expression levels of c-Myc and Cyclin D1 that had been inhibited by HNA, suggesting a crucial role of miR-34a in the IRE1α-mediated stress response pathway. Taken together, our results strongly suggest that targeting the IRE1α driven pro-survival pathways is an exciting therapeutic approach for the treatment of AML. Note: This abstract was not presented at the meeting. Citation Format: Haibo Sun, Behzad Kharabi Masouleh, Sigal Gery, Qi Cao, Serhan Alkan, Takayuki Ikezoe, Chie Akiba, Ronald Paquette, Wenwen Chien, Carsten Müller-Tidow, Yang Jing, Kharabi Masouleh, Markus Müschen, H. Phillip Koeffler. Inhibition of IRE1α-driven pro-survival pathways is a promising therapeutic application in acute myeloid leukemia. [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 510. doi:10.1158/1538-7445.AM2014-510

Expression of c-myc, c-myb and c-erbB-2 protein in breast cancer

Objective To investigate the roles of aberrant expression of c-myc,c-myb,and c-erbB-2 in the development of breast cancer and its clinical significance.Methods Immunohistochimical technique (S-P method)was used to detect the expression of c-myb,c-myc and c-erbB-2 protein in 150 cases of breast cancer and 30 cases of benign proliferative lesions.heir relation to clinical pathological parameters were analyzed.Results The protein expression levels of c-myc,c-myb,and c-erbB-2 in breast cancer were higher than those in benign proliferative lesions and the difference was significant(all P ＜0.05).Expression of c-myc protein had obvious relationship to histological grade,lymph nodes metastasis and pathological types(all P ＜ 0.05).Expression of c-myb protein related significantly to histological grade (P ＜ 0.05).c-erbB-2 was associated with the tumor size,grading,PR expression (all P ＜ 0.05).The association among the three kinds of genetic proteins was observed.Conclusion Expressions of oncogenes c-myc,c-myb,and c-erbB-2 proteins are higher in breast cancer and lower in benign proliferative lesions.The difference is significant.They are related to the grading and some other prognostic factors such as tumor size,pathologic types,PR expression and lymph node metastasis.The results suggest that these oncogenes are activated in the development of breast cancer and provoke cancerous cells multiplication.Expressions of oncogenes c-myc,c-myb and c-erbB-2 mightbe considered to be the prognostic indicators of breast cancer. Key words: Breast cancer; C-myb; C-myc; C-erbB-2; Pathology; Immunohistochemistry