Soft Agar Colony Formation Assay Research Articles

Abstract 3340: Endogenous, CRISPR-mediated overexpression of MYC family members as a framework to discover MYC-specific vulnerabilities

Abstract MYC activation reshapes cellular transcription, promotes cell growth, cell cycle progression and transformation and therefore MYC is considered a strong driver of tumor development. The MYC family includes c-MYC, MYCN, and MYCL that are typically associated with distinct tumor types. Based on the structural properties of these transcription factors MYC family members are considered undruggable targets. However, potential vulnerabilities associated with the cellular MYC status may provide a basis for therapeutic intervention. Currently, there is a lack of comparative studies assessing the specific contribution of each MYC family member to tumor development. Established cell lines in which MYC family members are overexpressed differ greatly in their genetic background, thereby impeding the analysis of MYC family member specific effects. Furthermore, the complexity of MYC regulation and MYC biology is not adequately recapitulated by traditional, heterologous MYC overexpression approaches. Therefore, we set out to establish an endogenous overexpression system using a CRISPR activation (CRISPRa) approach to activate the individual MYC family members for a systematic comparison of their impact on target gene activation, cell growth and drug sensitivity. Using sgRNAs specific to the promoter regions of the particular MYC family members in combination with a nucleolytically inactive Cas9-VP64 fusion protein, we successfully achieved transcriptional upregulation of all three MYC family members in multiple cell lines including NIH3T3 and MEF cells. The increase in MYC mRNA resulted in markedly elevated MYC protein levels. In addition, MYC activation enhanced transcription of canonical MYC target genes like NPM1, FBL, and 45S pre-rRNA suggesting a functional impact of upregulated MYC. Moreover, activation of c-Myc led to increased anchor-independent growth of cells in a soft-agar colony formation assay indicating that high c-Myc levels induce transformation in contrast to N-Myc or L-Myc. In line with previous studies that implicated upregulated c-Myc as an initiator of genome instability, we observed elevated levels of DNA damage by monitoring phosphorylated histone variant H2AX (γH2AX) in cells with activated c-Myc whereas N-Myc or L-Myc failed to induce γH2AX. Activation of c-Myc further led to increased sensitivity to cisplatin and other chemotherapeutic agents. In conclusion, we established an endogenous MYC overexpression model system that enables a systematic comparison of the different MYC family members and their specific contribution to tumor initiation, growth and drug sensitivity. The system successfully recapitulated previous observations of MYC activation and can serve as a platform to probe for MYC family member specific vulnerabilities and thereby pave the way for novel, targeted therapy approaches in MYC-driven cancer. Citation Format: Marcel A. Dammert, Martin L. Sos. Endogenous, CRISPR-mediated overexpression of MYC family members as a framework to discover MYC-specific vulnerabilities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3340.

Effects of MnSOD silence on in vitro tumorigenicity in NCI-H446 cells

To investigate the effect of manganese superoxide dismutase (MnSOD) silence on the in vitro tumorigenicity in human small cell lung cancer NCI-H446 cells and the underlying mechanisms. Methods: Sphere formation cells from NCI-H446 cells were obtained by suspension culture, while the expression of MnSOD and urokinase type plasminogen activator (uPAR) was analyzed by Western blot. Silence of MnSOD was performed by adenovirus infection in the second passage formation cells, and the effect of MnSOD silence on tumorigenicity in NCI-H446 cells was evaluated by sphere formation assay and soft-agar colony formation assay, while the expression of uPAR was analyzed by Western blot. Results: Compared with NCI-H446 cells, the sphere formation rate, colony formation rate, and the expression of MnSOD and uPAR were significantly increased in the second passage sphere formation cells in NCI-H446 cells (P<0.05). Silence of MnSOD inhibited the sphere formation rate, colony formation rate, and the expression level of uPAR in the second passage sphere formation cells in NCI-H446 cells. Conclusion: MnSOD may promote tumorigenicity in NCI-H446 cells by up-regulation of uPAR expression in vitro.

Synergic Inhibition of Lung Carcinoma 95-D Cell Proliferation and Invasion by Combination with (−)-Epigallocatechin-3-Gallate and Ascorbic Acid

In this study, the anti-invasion effects of (−)-epigallocatechin- 3-gallate (EGCG) mixed with ascorbic acid (Vc) on human lung carcinoma 95-D cells in vitro were examined and the synergism of the combination of EGCG and Vc was evaluated. Soft agar colony formation assay, cell migration assay, invasion assay, western blot analysis of NF-κB, in situ detection of cellular oxidative stress, and statistical analysis were assessed. The results showed that combining EGCG with Vc could inhibit clone forming rate of 95-D cell by 73.2%, reduce the migration ability of 95-D cell by 65.9%, and decrease the intracellular reactive oxygen species(ROS) level by 76.8%. The results of western blot proved that Vc enhanced the activity of EGCG in inhibiting NF-κB localization. It is speculated that the combination of EGCG and Vc can strongly suppress the proliferation and metastasis of lung carcinoma cells in a synergic manner, possibly with a mechanism associated with the scavenging of reactive oxygen species.

MiR-514 attenuates proliferation and increases chemoresistance by targeting ATP binding cassette subfamily in ovarian cancer.

Cisplatin is one of the most popular chemotherapeutic drugs in treating ovarian cancer. Resistance to cisplatin is a common clinical challenge that needs to be solved to increase its anti-tumor effects. The relation of miR-514 expression with prognosis in ovarian cancer patients was analyzed based on GSE73584 datasets. The regulation of miR-514 on proliferation and cisplatin chemosensitivity of ovarian cells was examined by MTT assay, colony-formation assay and soft-agar colony-formation assay. Dual luciferase assay was performed to detect the direct interaction of miR-514 with its downstream targets. Immunobloting and qRT-PCR were performed for target gene expression analysis. Low expression of miR-514 was related to poor prognosis in ovarian cancer patients. MiR-514 repressed proliferation and decreased cisplatin chemosensitivity in ovarian cancer cells by targeting ATP binding cassette subfamily. MiR-514 is of clinically significance in ovarian cancer by attenuating proliferation of ovarian cancer cells and decreasing chemoresistance of cisplatin by targeting ATP binding cassette subfamily.

Mycoplasma hyorhinis reduces sensitivity of human lung carcinoma cells to Nutlin-3 and promotes their malignant phenotype.

MDM2 inhibitors are promising anticancer agents that induce cell cycle arrest and tumor cells death via p53 reactivation. We examined the influence of Mycoplasma hyorhinis infection on sensitivity of human lung carcinoma cells NCI-H292 to MDM2 inhibitor Nutlin-3. In order to unveil possible mechanisms underlying the revealed effect, we investigated gene expression changes and signal transduction networks activated in NCI-H292 cells in response to mycoplasma infection. Sensitivity of NCI-Н292 cells to Nutlin-3 was estimated by resazurin-based cell viability assay. Genome-wide transcriptional profiles of NCI-H292 and NCI-Н292Myc.h cell lines were determined using Illumina Human HT-12 v3 Expression BeadChip. Search for key transcription factors and key node molecules was performed using the geneXplain platform. Ability for anchorage-independent growth was tested by soft agar colony formation assay. NCI-Н292Myc.h cells were shown to be 1.5- and 5.2-fold more resistant to killing by Nutlin-3at concentrations of 15 and 30µM than uninfected NCI-Н292 cells (P < 0.05 and P < 0.001, respectively). Transcriptome analysis revealed differential expression of multiple genes involved in cancer progression and metastasis as well as epithelial-mesenchymal transition (EMT). Moreover, we have shown experimentally that NCI-Н292Myc.h cells were more capable of growing and dividing without binding to a substrate. The most likely mechanism explaining the observed changes was found to be TLR4- and IL-1b-mediated activation of NF-κB pathway. Our results provide evidence that mycoplasma infection is an important factor modulating the effect of MDM2 inhibitors on cancer cells and is able to induce EMT-related changes.

MiRNA-34a decreases ovarian cancer cell proliferation and chemoresistance by targeting HDAC1.

This study aimed to explore the roles of miRNA-34a (miR-34a) in ovarian cancer (OC) cells and uncover possible mechanisms. The proliferation of OC cells was measured with an MTT assay and soft agar colony formation assay. TargetScan analysis, real-time PCR, and a luciferase reporter assay were used to demonstrate the downstream target of miR-34a in OC cells. HDAC1 expression levels were detected by immunoblot analysis. miR-34a inhibited the proliferation of SKOV3 and OVCA433 cells and enhanced cisplatin sensitivity in cisplatin-resistant SKOV3cp cells. The results of TargetScan analysis, real-time PCR, and luciferase reporter assay confirmed that miR-34a downregulated HDAC1 expression by directly targeting the 3'-UTR of HDAC1 mRNA. The overexpression of HDAC1 decreased cisplatin sensitivity and promoted proliferation in OC cells. MTT assay and soft agar colony formation assay showed that HDAC1 overexpression blocked the suppressive effects of miR-34a on SKOV3 cell proliferation. In addition, treatment with the miR-34a mimic partially recovered the cisplatin sensitivity of SKOV3cp cells, whereas HDAC1 overexpression blocked the above phenomena caused by treatment with the miR-34a mimic. miR-34a exhibited suppressive effects on OC cells via directly binding and downregulating HDAC1 expression, which subsequently decreased the resistance to cisplatin and suppressed proliferation in OC cells.

N-methyl-N-nitro-N-nitrosoguanidine-mediated ING4 downregulation contributed to the angiogenesis of transformed human gastric epithelial cells

AimsAngiogenesis is associated with the progression and mortality of gastric cancer. Epidemiological evidences indicate that long-term N-nitroso compounds (NOCs) exposure predominantly contributes to the mortality of gastric cancer. Therefore, further reduced mortality of gastric cancer demands to explore the exact mechanisms of NOCs induced angiogenesis. As a tumor suppressor gene, inhibitor of growth protein 4 (ING4) plays an important role in pathological angiogenesis. In this study, we will investigate ING4 expression level in human gastric epithelial cells after the long-term low dose exposure of N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and the pathological impact of MNNG-reduced ING4 on angiogenesis of transformed cells. Main methodsThe soft agar colony formation assay, Western blotting, immunofluorescence and wound healing assay were used to evaluate the characteristics of transformed cells. HUVEC growth and tube formation assays were performed to test the angiogenic abilities. EMSA, luciferase reporter gene assay, real-time PCR and Western blotting were used to explore the exact mechanism. Key findingsBy establishing transformed human gastric epithelial cells via chronic low dose treatment, a gradually ING4 downregulation was observed in the later-stage of MNNG-induced cell transformation. Moreover, we demonstrated that MNNG exposure-reduced ING4 expression significantly resulted into aggravating angiogenesis through increasing the phosphorylation level of NF-κB p65 and subsequently DAN binding activity and regulating the expressions of NF-κB p65 downstream pro-angiogenic genes, MMP-2 and MMP-9. SignificanceOur findings provided a significant mechanistic insight into angiogenesis of MNNG-transformed human gastric epithelial cell and supported the concept that ING4 may be a relevant therapeutic target for gastric cancer.

High expression of hnRNPA1 promotes cell invasion by inducing EMT in gastric cancer

Advanced gastric cancer (GC) has a poor prognosis and its treatment strategies are not very efficient. Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) has emerged as a plausible GC marker, however the role and molecular mechanism of hnRNPA1 in cell invasion and migration remains unknown. In the present study, the gene expression across normal and tumor tissue (GENT) database was used to evaluate the mRNA expression of hnRNPA1 in various types of cancer. Western blot analysis (WB) and immunohistochemistry (IHC) were performed to detect the protein expression of hnRNPA1 in GC tissues and adjacent non-tumor tissues. The expression of multiple oncogenes was detected by western blot analysis and quantitative RT-PCR in hnRNPA1 overexpressing GC cells. Soft agar colony formation, EdU incorporation, wound healing and invasion assays were applied to verify the role of hnRNPA1 in anchorage-independent cell growth, migration and invasion in GC cells. Epithelial-to-mesenchymal transition (EMT) markers were detected by immunofluorescence, western blot analysis and IHC in vitro. A nude mice model of metastasis carcinoma was established to confirm the role of hnRNPA1 during EMT in vivo. Our results revealed that hnRNPA1 was significantly upregulated in GC tissue. HnRNPA1 overexpression significantly induced cell growth, migration and invasion ability in GC cells. In addition, hnRNPA1 promoted EMT of GC cells in vitro and in vivo. These findings indicated that hnRNPA1 is highly expressed in GC and promoted invasion by inducing EMT transition in GC cells. Thus, hnRNPA1 may be a potential therapeutic target for GC.

Inhibition of miR-155, a therapeutic target for breast cancer, prevented in cancer stem cell formation.

Breast cancer is a common cancer in women of worldwide. Cancer cells with stem-like properties played important roles in breast cancer, such as relapse, metastasis and treatment resistance. Micro-RNA-155 (miR-155) is a well-known oncogenic miRNA overexpressed in many human cancers. The expression levels of miR-155 in 38 pairs of cancer tissues and adjacent normal tissues from breast cancer patients were detected using quantitative real-time PCR. The invasive cell line MDA-MB-231 was used to quantify the expression of miR-155 by tumor-sphere forming experiment. Soft agar colony formation assay and tumor xenografts was used to explore whether the inhibition of miR-155 could reduce proliferation of cancer cells in vivo and vitro. In the study, we found miR-155 was upregulated in BC. Soft agar colony formation assay and tumor xenografts showed inhibition of miR-155 could significantly reduce proliferation of cancer cells in vivo and vitro, which confirmed that miR-155 is an effective therapeutic target of breast cancer. Sphere-forming experiment showed that overexpression of miR-155 significantly correlated with stem-like properties. Expressions of ABCG2, CD44 and CD90 were repressed by inhibition of miR-155, but CD24 was promoted. Interestingly, inhibition of miR-155 rendered MDA-MB-231 cells more sensitive to Doxorubicinol, which resulted in an increase of inhibition rate from 20.23% to 68.72%. Expression of miR-155 not only was a therapeutic target but also was associated with cancer stem cell formation and Doxorubicinol sensitivity. Our results underscore the importance of miR-155 as a therapeutic target and combination of Doxorubicinol and miR-155-silencing would be a potential way to cure breast cancer.

MiR-192 and miR-662 enhance chemoresistance and invasiveness of squamous cell lung carcinoma

ObjectivesOverexpression of miR-192, miR-192* and miR-662 was previously found to correlate with poor prognosis of early-stage squamous cell lung cancer (SCC) patients. In this study, we investigated the relevance of these miRNAs to cancer cell biology and chemoresistance. Materials and methodsMiRNA expression profile was analysed in 10 non-small cell lung cancer (NSCLC) cell lines using RT-qPCR. H520 and H1703 cells were transfected with miRNA inhibitors (anti-miR-192, -192* and -662) for functional studies. Chemoresistance to cisplatin and etoposide was evaluated using MTT colorimetric assay. H520 cells were subjected to 3D soft-agar colony formation assay and H1703 cells to wound healing assay. Whole transcriptome analysis was used to assess the effect of miR-192 and miR-662 inhibition on gene expression. ResultsSCC cell lines, H520 and H1703, differed in miRNA expression and phenotypic features. MiR-192 and miR-662 inhibition decreased clonogenicity and motility of SCC cells. MiR-192 and miR-662 inhibition sensitized SCC cells to etoposide but not to cisplatin. Whole transcriptome analysis revealed genes regulated by miR-192 and miR-662 in SCC, relevant to maintaining chemoresistance, invasiveness, epithelial-mesenchymal transition (EMT) and immune evasion. ConclusionsWe showed for the first time that miR-192 and miR-662 have functional role in SCC cells. Our findings suggest that targeting these miRNAs may impact both chemoresistance and invasiveness of SCC, and add to the evidence linking these aspects of tumour biology. Overexpression of miR-192 and miR-662 might be useful as a marker of resistance to etoposide.

Acetonic extracts of the endolichenic fungus EL002332 isolated from Endocarpon pusillum exhibits anticancer activity in human gastric cancer cells

BackgroundEndolichenic fungi are microbes that inhabit the thalli of lichens and produce various unique chemicals that can be used for pharmaceutical purposes. PurposeThis study screened a library of endolichenic fungal extracts to identify novel anticancer agents capable of suppressing the tumorigenicity of human cancer cells. MethodsActive compounds were isolated from extracts of endolichenic fungi by column chromatography and reverse-phase HPLC. The anticancer effects of the extracts on cell viability was assessed with the use of MTT assay, Western blotting, fluorescence labeling of apoptotic cell, and flow cytometric analysis; and cell motility with the use of migration, invasion and soft agar colony-formation assay in vitro; and on skin and intraperitoneal mouse xenograft tumors in vivo were investigated. The therapeutic effects of the extract alone or in combination with the conventional chemoreagent docetaxel were analyzed by sulforhodamine B assay. ResultsAcetone extracts of EL002332, isolated from Endocarpon pusillum collected in the China desert in 2010, showed selective cytotoxicity against AGS human gastric cancer cells and CT26 mouse colon cancer cells. An active pure compound named myC was isolated from mycelium acetone extracts in a liquid culture system and showed more potent cytotoxicity than crude extracts in the AGS cell line. Especially, myC greatly increased the apoptotic cell population at the IC50 concentration and activated apoptotic signaling by regulating Bcl2 family protein expression and caspase pathway activity. EL002332 crude extracts and myC decreased AGS cell motility at sub-lethal concentrations. In vivo skin and intraperitoneal xenograft tumor experiments showed that the size of tumors and the tumor score were significantly smaller in EL002332 crude extract-treated groups than in control groups. EL002332 crude extracts showed synergistic effects with docetaxel on the AGS and TMK1 cell lines. ConclusionThe endolichenic fungus EL002332 has potential anticancer activity in gastric cancer and peritoneal carcinomatosis.

Herbal formula YGJDSJ inhibits anchorage-independent growth and induces anoikis in hepatocellular carcinoma Bel-7402 cells

BackgroundBased on clinical medications and related studies, we established a Yang-Gan Jie-Du Sang-Jie (YGJDSJ) herbal formula for hepatocarcinoma treatment. In present study, we evaluated the anti-cancer potential of YGJDSJ on suspension-grown human hepatocellular carcinoma Bel-7402 cells.MethodsBel-7402 cells were cultured in poly(2-hydroxyethyl methacrylate) (poly-HEMA) coated plates and treated with YGJDSJ. Anchorage-independent cell growth was detected by cell Counting Kit-8 (CCK-8) assay and soft agar colony formation assay. Anoikis was detected by ethdium homodimer-1 (EthD-1) staining and flow cytometry analysis. Caspases activities were detected by the cleavage of chromogenic substrate. Reactive oxygen species (ROS) was detected by 2′,7′-dichlorofluorescin diacetate (DCFH-DA) staining. Protein expression and phosphorylation was identified by western blot. Protein expression was knocked-down by siRNA.ResultsYGJDSJ inhibited the proliferation of Bel-7402 cells in poly-HEMA coated plates and anchorage-independent growth of Bel-7402 cells in soft agar. YGJDSJ also induced anoikis in Bel-7402 cells as indicated by EthD-1 staining and flow cytometry analysis. YGJDSJ activated caspase-3, − 8, and − 9 in suspension-grown Bel-7402 cells. The pan-caspase inhibitor Z-VAD-FMK significantly abrogated the effects of YGJDSJ on anoikis in suspension-grown Bel-7402 cells. In addition, YGJDSJ increased ROS in suspension-grown Bel-7402 cells. The ROS scavenger N-acetyl-L-cysteine (NAC) partially attenuated YGJDSJ-induced activation of caspase-3, − 8 and − 9 and anoikis in suspension-grown Bel-7402 cells. Furthermore, YGJDSJ inhibited expression and phosphorylation of protein tyrosine kinase 2 (PTK2) in suspension-grown Bel-7402 cells. Over-expression of PTK2 significantly abrogated YGJDSJ induced anoikis.ConclusionsYGJDSJ inhibits anchorage-independent growth and induce caspase-mediated anoikis in Bel-7402 cells, and may relate to ROS generation and PTK2 downregulation.

Retracted] Effects of cyclin E gene silencing on the proliferation of esophageal cancer cell lines, EC9706, Eca109 and KYSE30.

Subsequently to the publication of this article, an interested reader drew to our attention the fact that the six panels shown in Fig.6 shared several areas of identity among them. Following an internal investigation, a laboratory technician, who was responsible for editing the pictures, admitted that the data as presented in the figure had been manipulated after having mislaid some of the original data. The corresponding author of the article takes responsibility for this oversight, and therefore the paper is to be retracted from publication. All of the named authors agree to this retraction. We deeply regret that these errors were allowed to remain in the paper, and extend our apologies to the readership of the Journal. [the original article was published in Molecular Medicine Reports 7: 799-804, 2013; DOI: 10.3892/mmr.2013.1280].

HUWE1 controls the development of non-small cell lung cancer through down-regulation of p53.

Lung cancer is the most frequent cancer type and the leading cause of tumor-associated deaths worldwide. TP53 is an important tumor suppressor gene and is frequently inactivated in lung cancer. E3 ligases targeting p53, such as MDM2, are involved in the development of lung cancer. The E3 ligase HUWE1, which targets many tumor-associated proteins including p53, has been reported to be highly expressed in lung cancer; however, its role in lung tumorigenesis is unclear.Methods: The expression of HUWE1 and p53 in lung cancer cells was modulated and the phenotypes were assessed by performing soft agar colony forming assays, cell cycle analysis, BrdU incorporation assays, and xenograft tumor growth assays. The effect on tumorigenesis in genetically-engineered mice was also analyzed. The mechanism through which HUWE1 sustained lung cancer cell malignancy was confirmed by western blotting. HUWE1 expression in clinical lung cancer was identified by immunohistochemistry and validated by analyzing lung adenocarcinoma and lung squamous carcinoma samples from the Cancer Genome Atlas (TCGA) database. Finally, we assessed the association between HUWE1 expression and patient outcome using online survival analysis software including survival information from the caBIG, GEO, and TCGA database.Results: Inactivation of HUWE1 in a human lung cancer cell line inhibited proliferation, colony-forming capacity, and tumorigenicity. Mechanistically, this phenotype was driven by increased p53, which was due to attenuated proteasomal degradation by HUWE1. Up-regulation of p53 inhibited cancer cell malignancy, mainly through the induction of p21 expression and the down-regulation of HIF1α. Huwe1 deletion completely abolished the development of EGFRVIII-induced lung cancer in Huwe1 conditional knockout mice. Furthermore, survival analysis of lung cancer patients showed that increased HUWE1 expression is significantly associated with worse prognosis.Conclusion: Our data suggest that HUWE1 plays a critical role in lung cancer and that the HUWE1-p53 axis might be a potential target for lung cancer therapy.

CHK2 Promotes Anoikis and is Associated with the Progression of Papillary Thyroid Cancer

Background/Aims: Cell cycle checkpoint kinase 2 (CHK2) performs essential cellular functions and might be associated with tumorigenesis and tumor progression. Here, we explored the function and molecular mechanisms of CHK2 in the progression of papillary thyroid cancer (PTC). Methods: The expression levels of both total CHK2 and activated CHK2 (p-CHK2) in tissues from 100 PTC patients were detected and evaluated using immunohistochemistry. The roles of CHK2 on cell proliferation, invasion, migration, apoptosis and cancer stem cell (CSC) markers were investigated by CCK-8, Transwell, flow cytometry, western blot and ALDEFLOUR assay. PTC cells cultured in suspension conditions were assayed for anoikis. The anchorage-independent condition was further detected by soft agar colony formation assay. Furthermore, anoikis associated regulatory proteins were explored by western blot and verified by forced downregulation experiment, respectively. Results: We found that the levels of both CHK2 and p-CHK2 were significantly upregulated in PTC cancer tissues compared with those in tumor-adjacent tissues. The overexpression of p-CHK2 in primary tumor tissues was associated with tumor aggressiveness and metastatic potential. However, the levels of both CHK2 and p-CHK2 were decreased in metastatic lymph nodes. Our results showed that CHK2 upregulated the levels of CSC markers with no effect on cell proliferation, invasion and migration. Interestingly, we revealed a previously undescribed anoikis-promoting role for CHK2 in PTC. Specifically, the detachment of PTC cells from the extracellular matrix (ECM) triggers CHK2 degradation. Then, the forced downregulation of CHK2 rescued PTC cells from anoikis, but no effect was observed on the apoptosis of adherent PTC cells. Additionally, as a novel regulator of anoikis, CHK2 can induce cell death in a p53-independent manner via the regulation of PRAS40 activation. Conclusion: High expression levels of CHK2 and p-CHK2 were associated with the progression of PTC. Our results defined an unexpected role for CHK2 as a mediator of anoikis that functions through the regulation of PRAS40 activation, which may be associated with the survival of circulating tumor cells and metastatic behavior.

Sumoylation of EphB1 Suppresses Neuroblastoma Tumorigenesis via Inhibiting PKCγ Activation

Background/Aims: An increasing number of studies have linked erythropoietin-producing hepatocellular carcinoma (Eph) family receptor tyrosine kinases to cancer progression. However, little knowledge is available about the regulation of their functions in cancer. Methods: SUMOylation was analyzed by performing Ni2+-NTA pull-down assay and immunoprecipitation. Cell proliferation, anchorage-independent growth, and tumorigenesis in vivo were examined by cell counting kit-8, soft agar colony formation assay, and a xenograft tumor mouse model, respectively. Results: We found that EphB1 was post-translationally modified by the small ubiquitin-like modifier (SUMO) protein at lysine residue 785. Analysis of wild-type EphB1 and SUMOylation-deficient EphB1 K785R mutant revealed that SUMOylation of EphB1 suppressed cell proliferation, anchorage-independent cell growth, and xenograft tumor growth. Mechanistic study showed that SUMOylation of EphB1 repressed activation of its downstream signaling molecule PKCγ, and consequently inhibited tumorigenesis. A reciprocal regulatory loop between PKCγ and SUMOylation of EphB1 was also characterized. Conclusion: Our findings identify SUMO1 as a novel key regulator of EphB1-mediated tumorigenesis.

Inhibition of Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Zeta (YWHAZ) Overcomes Drug Resistance and Tumorigenicity in Ovarian Cancer

Background/Aims: Cancer stem-like cells are the main cause of tumor occurrence, progression, and therapeutic resistance. However, the precise signals required for the maintenance of the stem-like traits of these cells in ovarian cancer remain elusive. We have thus worked to elucidate the functional role of Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ), a gene encoding the 14-3-3ζ protein, in the regulation of multidrug resistance and stem cell-like traits in ovarian cancer. Methods: We detected the YWHAZ levels in human ovarian cancer specimens and cell lines using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blots. MTS assays, soft agar colony formation assays, migration assays, cell cycle analysis, sphere formation assays, and flow cytometry were applied to investigate the functional role of YWHAZ in ovarian cancer. Results: Our data reveals substantially increased YWHAZ expression in both cisplatin- and paclitaxel-resistant ovarian cancer cells. Silencing YWHAZ restored the sensitivity of resistant ovarian cancer cells to cisplatin and paclitaxel. Furthermore, in vitro studies showed that down-regulation of YWHAZ inhibited cell cycle progression, migration, and the expression of stem cell markers. Moreover, tumorigenicity was suppressed in tumor-bearing BALB/c nude mice following YWHAZ knockdown. Additionally, we demonstrated that the expression of YWHAZ was directly down-regulated by miR-30e in resistant ovarian cancer cells. Conclusion: Our results have led to new insights into the essential role of YWHAZ in the regulation of tumourigenesis, stem-like traits, and drug resistance in ovarian cancer, thereby helping to identify a potential target for ovarian cancer therapy.

Exploring the potential of andrographolide and its derivatives as anti-pancreatic cancer therapeutics: in silico, in vitro, and in vivo approaches

Event Abstract Back to Event Exploring the potential of andrographolide and its derivatives as anti-pancreatic cancer therapeutics: in silico, in vitro, and in vivo approaches Shun Ying Quah1, Kok Lian Ho1, Nizar Abdul Manan1, Sreenivasa R. Sagineedu2, Pran Kishore Deb3 and Johnson Stanslas1* 1 Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Malaysia 2 International Medical University, Malaysia 3 Philadelphia University, Jordan Background Approximately 95% of pancreatic ductal adenocarcinoma (PDAC) patients are found having oncogenic K-Ras, which perturbs mitogen-activated protein kinase (MAPK) cascade critical for cellular processes. Our previous analysis identified andrographolide (AGP) derivatives, SRJ09 and SRJ23, disrupted MAPK activation by binding to K-Ras. Chemical modification of SRJ23 produced S8, which selectively inhibited PDAC cell growth. This study aims to evaluate these compounds computationally on K-Ras, and their in vitro and in vivo anticancer activity against PDAC. Methods Using Glide (Schrödinger, LLC), molecular docking was performed to identify the binding mode and affinity of AGP and its analogues to wild-type and mutant K-Ras. PANC-1 cells harbouring K-RasG12D, the most predominant K-Ras mutation, were used in cell-based and animal studies. Soft-agar colony formation assay was performed to determine the ability of compounds to inhibit anchorage-independent cell growth. GTP-loading of K-Ras and MAPK activation were assessed by immunoblotting. Anti-tumour activity was assessed in athymic nude mice carrying PANC-1 tumour xenografts. Tumour tissues were evaluated histologically by haematoxylin-eosin staining. Results S8 bound stronger to K-Ras mutants, specifically K-RasG12D and K-RasG12V (-76.54kcal/mol and -80.43kcal/mol, respectively, versus -69.40kcal/mol in wild-type K-Ras). Contrary to AGP and SRJ09, SRJ23 and S8 anchored into a binding pocket located between two switches of K-RasG12V, via their lactone ring, where the 14-OH group interacted with aspartate-54 through hydrogen-bonding. SRJ23 and S8 comparably inhibited PANC-1 colony formation in a dose-dependent manner. However, S8 produced greater repression in GTP-loading of K-Ras and suppressed Erk phosphorylation in PANC-1 cells. S8 significantly decelerated tumour growth in PANC-1-xenografted nude mice at a dose of 100mg/kg, and caused a higher degree of tumour necrosis, in comparison to SRJ23 treatment at the same dose. Conclusion S8 likely binds to oncogenic K-Ras, leading to inhibition of the aberrant Ras-MAPK signalling and reduction in pancreatic tumourigenesis in vitro and in vivo. Keywords: PDAC, MAPK, tumourigenesis, Andrographolide derivatives, Oncogenic K-Ras Conference: International Conference on Drug Discovery and Translational Medicine 2018 (ICDDTM '18) “Seizing Opportunities and Addressing Challenges of Precision Medicine”, Putrajaya, Malaysia, 3 Dec - 5 Feb, 2019. Presentation Type: Oral Presentation Topic: Cancer Citation: Quah S, Ho K, Abdul Manan N, Sagineedu SR, Deb P and Stanslas J (2019). Exploring the potential of andrographolide and its derivatives as anti-pancreatic cancer therapeutics: in silico, in vitro, and in vivo approaches. Front. Pharmacol. Conference Abstract: International Conference on Drug Discovery and Translational Medicine 2018 (ICDDTM '18) “Seizing Opportunities and Addressing Challenges of Precision Medicine”. doi: 10.3389/conf.fphar.2018.63.00022 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 30 Sep 2018; Published Online: 17 Jan 2019. * Correspondence: Prof. Johnson Stanslas, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Malaysia, rcxjs@upm.edu.my Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Shun Ying Quah Kok Lian Ho Nizar Abdul Manan Sreenivasa R Sagineedu Pran Kishore Deb Johnson Stanslas Google Shun Ying Quah Kok Lian Ho Nizar Abdul Manan Sreenivasa R Sagineedu Pran Kishore Deb Johnson Stanslas Google Scholar Shun Ying Quah Kok Lian Ho Nizar Abdul Manan Sreenivasa R Sagineedu Pran Kishore Deb Johnson Stanslas PubMed Shun Ying Quah Kok Lian Ho Nizar Abdul Manan Sreenivasa R Sagineedu Pran Kishore Deb Johnson Stanslas Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

The RNA-Binding Protein PCBP1 Functions as a Tumor Suppressor in Prostate Cancer by Inhibiting Mitogen Activated Protein Kinase 1

Background/Aims: Poly r(C) binding protein (PCBP) 1 or heterogeneous ribonucleoprotein (hnRNP) E1 is a RNA binding protein functional in multiple biological processes. In prostate cancer (PCa), PCBP1 loss was shown to be involved with increased stemness in PCacells; however, the underlying mechanism remains unclear. Method: The role of PCBP1 in prostate tumor formationwas determined by xenograft assays. Immunoprecipitationand mass spectrometry were performed to find the pathways altered after PCBP1 knockdown. Cell proliferation, migration, invasion, and soft agar colony formationassays and xenograft assays were used to determine the role of target protein pathogenesis regulation and formation of PCa. QRT-PCR was performedto quantify relative mRNA expression. Results: The expression of mitogen activated protein kinase 1 (MAPK1) or extracellular signal regulated kinase 2 (ERK2) was increased following PCBP1 loss. Attenuation of MAPK1 inhibited in vitro and in vivo tumorigenicity and metastasis in PCa cell line, PC3. Overexpression of MAPK1 in the PC3 cells increased the tumorigenicity and metastasis. Analysis of PCBP1 and MAPK1 mRNA levels in 25 PCa patients compared to tumor-adjacent normal tissue confirmed an inverse correlation between PCBP1 and MAPK1 expression. Conclusions: PCBP1 can act as a suppressor of tumor in prostate epithelial cells by inhibiting MAPK1 expression.

Role of Glypican-3 in the growth, migration and invasion of primary hepatocytes isolated from patients with hepatocellular carcinoma.

Recently, Glypican-3 (GPC3) has been identified as a potential hepatocellular carcinoma (HCC) diagnostic and/or therapeutic target. GPC3 has been found to be up-regulated in HCC and to be absent in normal and cirrhotic liver. As yet, however, the molecular characteristics of GPC3 and its role in HCC cell physiology and development are still undefined. Human hepatocyte cultures were established from 10 HCC patients. Additional liver samples were obtained from 5 patients without cirrhosis and/or HCC. Soft agar colony formation, (co-)immunofluorescence and Western blot assays were used to characterize the hapatocyte cultures. The expression of GPC3 in the hepatocytes was silenced using siRNA, after which, apoptosis, scratch wound migration and transwell invasion assays were performed. We found that in HCC precursor hepatocytes GPC3 is increasingly expressed in different forms and at different locations, i.e., a non-cleaved form (70kDa) was found to be localized in the cytoplasm while a N-terminal cleaved form (N-GPC3: 40kDa) was fond to be localized in the cytoplasm and at the extracellular side of hepatocyte membranes. In addition, we found that the non-cleaved form of GPC3 co-localizes with Furin-Convertase in the Golgi apparatus. We also found that, similar to GPC3, Furin-Convertase is expressed in HCC precursor cells, suggesting a role in GPC3 processing. Subsequent siRNA-mediated GPC3 silencing resulted in a temporary inhibition of cell proliferation, migration and ivasion, while inducing apoptosis in transformed hepatocytes. Our data reveal new aspects of the role of GPC3 in early hepatocyte transformation. In addition we conclude that GPC3 may serve as a new HCC immune-therapeutic target.