Anchorage-independent Growth Ability Research Articles

MiR-524-5p reduces the progression of the BRAF inhibitor-resistant melanoma

BRAF inhibitors were approved for the treatment of BRAF-mutant melanoma. However, most patients acquire the resistance to BRAF inhibitors after several months of treatment. miR-524-5p is considered as a tumor suppressor in many cancers, including melanoma. In this study, we investigated the biological functions of miR-524-5p in melanoma with acquired resistance to BRAF inhibitor and evaluated the endogenous miR-524-5p expression as a biomarker for melanoma. The results showed that the expression of miR-524-5p was 0.481-fold lower in melanoma tissues (n = 117) than in nevus tissues (n = 40). Overexpression of miR-524-5p significantly reduced proliferative, anchorage-independent growth, migratory and invasive abilities of BRAF inhibitor-resistant melanoma cells. Moreover, the introduction of miR-524-5p led to a reduced development of BRAF inhibitor-resistant melanoma in vivo. Remarkably, the MAPK/ERK signaling pathway was decreased after treatment with miR-524-5p. Furthermore, next-generation sequencing analysis implied that the complement system, leukocyte extravasation, liver X receptor/retinoid-X-receptor activation, and cAMP-mediated signaling may be related to miR-524-5p-induced pathways in the resistant cells. The miR-524-5p level was higher on average in complete response and long-term partial response patients than in progressive disease and short-term partial response patients treated with BRAF inhibitors. Our results proposed that miR-524-5p could be considered as a target for treatment BRAF inhibitor-resistant melanoma and a prognostic marker in the response of patients to BRAF inhibitors for melanoma.

Abstract PO-130: The mitochondrial protein ATAD3A promotes cisplatin resistance in oral squamous cell carcinoma

Abstract Cisplatin is well-known for its damaging effect on nuclear DNA, leading to cell death due to blockage of DNA replication and transcription, but this activity cannot fully explain why cisplatin displays cytotoxicity in post-mitotic tissues, suggesting other mechanisms may be in play. Many studies have challenged the current model of cisplatin resistance, however, the precise mechanism behind is still unknown. Here, we report that the mitochondrial protein ATAD3A functions as an oncogene in oral squamous cell carcinoma (OSCC), promoting cell proliferation and enhancing the ability of colony formation and anchorage-independent growth. Moreover, ATAD3A is involved in cisplatin resistance in OSCC, and knockdown of ATAD3A enhanced the sensitivity of OSCC cells to cisplatin. It appears that ATAD3A-mediated cisplatin resistance is regulated by other mechanisms rather than its expression levels, as evidenced by overexpression of a Walker A (WA) dead mutant of ATAD3A leads to a suppression in OSCC cell growth and resensitizes cisplatin-resistant OSCC cells to this drug, indicating a potent dominant-negative effect of defective ATP-binding of ATAD3A in chemotherapy. Our study demonstrates for the first time that ATAD3A plays an oncogenic role in OSCC and abrogating its ATP-binding ability may represent a promising regimen for better treatment of patients with OSCC, especially for those who suffer from cisplatin-resistance. Citation Format: Liwei Lang, Leslie Duncan, Caleb Jensen, Chloe Shay, Yong Teng. The mitochondrial protein ATAD3A promotes cisplatin resistance in oral squamous cell carcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-130.

HDAC9 Is Preferentially Expressed in Dedifferentiated Hepatocellular Carcinoma Cells and Is Involved in an Anchorage-Independent Growth.

Simple SummaryHistone deacetylases (HDACs) are known to play a role in malignant transformation of cancer cells, however, the critical HDAC responsible for the dedifferentiation of hepatocellular carcinoma (HCC) cells remains unclear. The aim of our study was to identify the HDAC related to the dedifferentiation of HCC. We confirmed preferential expression of HDAC9, a class II HDAC, in undifferentiated hepatoma cells and a positive correlation of gene expression between HDAC9 and dedifferentiation markers by database analysis of HCC patients. Genetic and pharmacological inhibition of HDAC9 showed decreased cell proliferation and sphere-forming activity, which indicates an ability of anchorage-independent cell growth and self-renewal. HDAC9 suppression showed significant down-regulation of aldehyde dehydrogenase 1A3 (ALDH1A3), a stemness-related gene reported in several malignancies including HCC. We also confirmed that ALDH activity is required for the anchorage-independent cell growth of undifferentiated HCC cells. Inhibition of HDAC9 may be a therapeutic strategy for targeting dedifferentiated HCC cells with stemness features.Aberrant activation of histone deacetylases (HDACs) is one of the causes of tumor cell transformation in many types of cancer, however, the critical HDAC responsible for the malignant transformation remain unclear. To identify the HDAC related to the dedifferentiation of hepatocellular carcinoma (HCC) cells, we investigated the expression profile of HDACs in differentiated and undifferentiated hepatoma cells. We found that HDAC9, a member of the class II HDAC, is preferentially expressed in undifferentiated HCC cells. Analysis of 373 HCC patients in The Cancer Genome Atlas (TCGA) database revealed that the expression of HDAC9 mRNA positively correlated with the markers of mesenchymal phenotype and stemness, and conversely, negatively correlated with hepatic differentiation markers. HDAC9 was transcriptionally upregulated in epithelial–mesenchymal transition (EMT)-induced HCC cells treated with TGF-β. Genetic and pharmacological inhibition of HDAC9 in undifferentiated HCC cells showed decreased sphere-forming activity, which indicates an ability of anchorage-independent cell growth and self-renewal. We also showed that aldehyde dehydrogenase 1A3 (ALDH1A3) was downregulated in HDAC9-suppressing cells, and ALDH inhibitor disulfiram significantly decreased the sphere formation of undifferentiated HCC cells. Together, our data provide useful information for the development of HDAC9-specific inhibitors for the treatment of HCC progression.

CD109 mediates tumorigenicity and cancer aggressiveness via regulation of EGFR and STAT3 signalling in cervical squamous cell carcinoma

BackgroundCD109 was involved in the tumorigenesis and progression of various cancers via TGF-β1 signalling and STAT3 activation. As CD109 is strongly expressed in cervical squamous cell carcinoma, this study was conducted to investigate its functional characteristics in cervical cancer.MethodsCD109 expression was examined by immunohistochemistry (IHC) with cervical tissue microarray. The effects of CD109 expression were examined on migration, cell proliferation, spheroid formation and soft-agar colony-formation assay. Meanwhile, cervical cancer cell lines with high CD109 expression were chosen for the functional study using siRNA knockdown and CRISPR/Cas9 knockout.ResultsIHC demonstrated an upregulation of CD109 in the cell membrane of cervical squamous cell carcinoma. CD109( + ) cells isolated by flow-cytometric sorting displayed enhanced migration, cell proliferation, sphere-forming and anchorage-independent cell growth ability. In contrast, silencing of CD109 expression could reverse the in vitro and in vivo tumorigenic and aggressive properties. Furthermore, CD109 induced EGFR-mediated STAT3 phosphorylation known to be responsible for cell migration, proliferation and maintenance of CSC phenotype.ConclusionAbundant CD109( + ) populations in cervical cancer cells potentially contributed to carcinogenesis and aggressiveness, whereas silencing of CD109 expression could reverse those properties. CD109 mediates cervical tumorigenicity and aggressiveness via CD109/EGFR/STAT3 signalling.

Neferine, an alkaloid from lotus seed embryo targets HeLa and SiHa cervical cancer cells via pro-oxidant anticancer mechanism.

Apoptosis and autophagy are important processes that control cellular homeostasis and have been highlighted as promising targets for novel anticancer drugs. This study aims to investigate the inhibitory effects and mechanisms of Neferine (Nef), an alkaloid from the lotus seed embryos of Nelumbo nucifera (N. nucifera), as a dual inducer of apoptosis and autophagy through the reactive oxygen species (ROS) activation in cervical cancer cells. Nef and N. nucifera extract suppressed the cell viability of HeLa and SiHa cells in a dose-dependent manner. Importantly, Nef showed minimal toxicity to normal cells. Furthermore, Nef inhibited anchorage-independent growth, colony formation and migration ability of cervical cancer cells. Nef induces mitochondrial apoptosis by increasing pro-apoptotic protein bax, cytochrome-c, cleaved caspase-3 and caspase-9, poly-ADP ribose polymerase (PARP) cleavage, DNA damage (pH2 AX) while downregulating Bcl-2, procaspase-3 and procaspase-9, and TCTP. Of note, apoptotic effect by Nef was significantly attenuated in the presence of N-acetylcysteine (NAC), suggesting pro-oxidant activity of this compound. Nef also promoted autophagy induction through increasing beclin-1, atg-4, atg-5 and atg-12, LC-3 activation, and P 62/SQSTM1 as determined by western blot analysis. Collectively, these results demonstrate that Nef is a potent anticancer compound against cervical cancer cells through inducing apoptosis and autophagic pathway involving ROS.

Highly Expressed FOXF1 Inhibit Non-Small-Cell Lung Cancer Growth via Inducing Tumor Suppressor and G1-Phase Cell-Cycle Arrest.

Cancer pathogenesis results from genetic alteration-induced high or low transcriptional programs, which become highly dependent on regulators of gene expression. However, their role in progressive regulation of non-small-cell lung cancer (NSCLC) and how these dependencies may offer opportunities for novel therapeutic options remain to be understood. Previously, we identified forkhead box F1 (FOXF1) as a reprogramming mediator which leads to stemnesss when mesenchymal stem cells fuse with lung cancer cells, and we now examine its effect on lung cancer through establishing lowly and highly expressing FOXF1 NSCLC engineered cell lines. Higher expression of FOXF1 was enabled in cell lines through lentiviral transduction, and their viability, proliferation, and anchorage-dependent growth was assessed. Flow cytometry and Western blot were used to analyze cellular percentage in cell-cycle phases and levels of cellular cyclins, respectively. In mice, tumorigenic behavior of FOXF1 was investigated. We found that FOXF1 was downregulated in lung cancer tissues and cancer cell lines. Cell proliferation and ability of migration, anchorage-independent growth, and transformation were inhibited in H441-FOXF1H and H1299-FOXF1H, with upregulated tumor suppressor p21 and suppressed cellular cyclins, leading to cell-cycle arrest at the gap 1 (G1) phase. H441-FOXF1H and H1299-FOXF1H injected mice showed reduced tumor size. Conclusively, highly expressing FOXF1 inhibited NSCLC growth via activating tumor suppressor p21 and G1 cell-cycle arrest, thus offering a potentially novel therapeutic strategy for lung cancer.

Exposure to low dose ZnO nanoparticles induces hyperproliferation and malignant transformation through activating the CXCR2/NF-κB/STAT3/ERK and AKT pathways in colonic mucosal cells

As ZnO nanoparticles have been applied in many fields, their biological risks on human health, of course, are worthy of our attention. Whether ZnO NPs have the risk and how colonic cells respond to the invaded ZnO NPs are still unknown. Herein, we evaluated the biological effects of ZnO NPs on colonic mucosal cells by in vitro and in vivo methods. IMCE cells, with APC mutation but phenotypically normal, demonstrated hyperproliferation through activating the CXCR2/NF-κB/STAT3/ERK and AKT pathways when exposed to ZnO NPs for 24 h. Long-term exposure of ZnO NPs resulted in the malignant transformation of IMCE cells, showing the morphological changes, anchorage-independent cell growth ability. Importantly, IMCE cells exposed to ZnO NPs subcutaneously grew and induced tumorigenesis in nude mice. In conclusion, exposure of ZnO NPs could induce malignant transformation of colonic mucosal cells through the CXCR2/NF-κB/STAT3/ERK and AKT pathways. We suggest that it was necessary to consider using the precautionary principle for gastrointestinal contact nanomaterials.

Suppression of Malignant Potentials of A549 Human Lung Cancer Cell Line by Downregulation of the β4-Galactosyltransferase 1 Gene Expression.

Our previous study demonstrated that downregulation of transcription factor Specificity protein (Sp) 1 suppresses the malignant potentials of A549 human lung cancer cell line with the reduced β4-galactosylation of highly branched N-glycans on cell surface glycoproteins. The reduced β4-galactosylation was brought about by the decreased expression of the β4-galactosyltransferase 1 (β4GalT1) gene. Herein, we examined whether the reduced β4-galactosylation by decreasing the β4GalT1 gene expression suppresses the malignant potentials of A549 cells. In the β4GalT1-downregulated cells, the β4-galactosylation of highly branched N-glycans was reduced in several glycoproteins such as lysosome-associated membrane protein-1 and E-cadherin. The anchorage-independent growth and migratory ability of the β4GalT1-downregulated cells decreased when compared with the control cells. Furthermore, the phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) decreased in the β4GalT1-downregulated cells. These results indicate that downregulation of the β4GalT1 gene decreases the β4-galactosylation of highly branched N-glycans and the phosphorylation of p44/42 MAPK, and suppresses the malignant potentials of A549 cells.

MicroRNA-527 Induces Proliferation and Cell Cycle in Esophageal Squamous Cell Carcinoma Cells by Repressing PH Domain Leucine-Rich-Repeats Protein Phosphatase 2.

Increasing evidence indicated that microRNAs served dominant roles in carcinogenesis and cancer progression by targeting potential downstream genes. In our study, we found that miR-527 was an upregulated expression in human esophageal squamous cell carcinoma (ESCC) cells and tissues. Furthermore, overexpression of miR-527 promoted cell proliferation and colony formation, enhanced anchorage-independent growth ability, and contributed to cell cycle. In addition, protein phosphatase 2 (PHLPP2) was identified as the direct downstream target gene of miR-527 and was confirmed by luciferase gene reporter assay. In summary, we concluded that miR-527 acted as an oncogenic microRNA in ESCC development by directly targeting PHLPP2 might be a novel therapeutic target for the treatment of ESCC.

E6AP Promotes a Metastatic Phenotype in Prostate Cancer

SummaryAlthough primary prostate cancer is largely curable, progression to metastatic disease is associated with very poor prognosis. E6AP is an E3 ubiquitin ligase and a transcriptional co-factor involved in normal prostate development. E6AP drives prostate cancer when overexpressed. Our study exposed a role for E6AP in the promotion of metastatic phenotype in prostate cells. We revealed that elevated levels of E6AP in primary prostate cancer correlate with regional metastasis and demonstrated that E6AP promotes acquisition of mesenchymal features, migration potential, and ability for anchorage-independent growth. We identified the metastasis suppressor NDRG1 as a target of E6AP and showed it is key in E6AP induction of mesenchymal phenotype. We showed that treatment of prostate cancer cells with pharmacological agents upregulated NDRG1 expression suppressed E6AP-induced cell migration. We propose that the E6AP-NDRG1 axis is an attractive therapeutic target for the treatment of E6AP-driven metastatic prostate cancer.

Forkhead box B2 inhibits the malignant characteristics of the pancreatic cancer cell line Panc-1 in vitro.

Forkhead box (FOX) proteins constitute a family of transcription factors that are evolutionarily conserved in various species ranging from yeast to humans. These proteins have functions during development as well as in adulthood. To date, many reports have described the functions of FOX family genes in cancer cells, but the role of FOXB2 is not well understood. In one of the pancreas ductal adenocarcinoma cell lines, Panc-1 cells, we showed here that FOXB2 expression is barely detectable and that CpG islands in the 5' regions of the FOXB2 are highly methylated. These findings led us to hypothesize that FOXB2 acts as a tumor suppressor. To clarify our hypotheses, we investigated the effects of FOXB2 over-expression in Panc-1 cells. We obtained FOXB2 stable transfectants, and these clones exhibited reduced spheroid formation ability. Expression of β-catenin, which is reported to be over-expressed in various cancer cells, was highly suppressed in FOXB2 stable transfectants. Moreover, side population (SP) cell fractions, which have a high tumorigenesis and metastatic potential, as well as anchorage-independent growth ability, were reduced. These results suggest that FOXB2 has the ability to inhibit the malignant characteristics of Panc-1 in vitro.

TCF19 contributes to cell proliferation of non-small cell lung cancer by inhibiting FOXO1.

Transcription factor 19 (TCF19) harbors a forkhead association (FHA) domain, a proline-rich region, a PHD or RING finger region, suggesting that TCF19 possesses a powerful function. However, its expression and function remains unknown in non-small-cell lung cancer (NSCLC). The function cluster analysis was carried out using Metascape website. 3-(4,5-Dimethyl-2-thiazolyl)2,5-diphenyl-2H-tetrazolium bromide (MTT), colony formation, and anchorage-independent growth ability assay were carried out to detect the effect of TCF19 on cell proliferation. Bromodeoxyuridine (Brdu) labeling and flow cytometry assay were used to evaluate the effect of TCF19 on cell-cycle progression. Quantitative polymerase chain reaction and chromatin immunoprecipitation assay were performed to investigate the mechanism by which TCF19 is involved in cell-cycle transition. By analyzing the publicly available dataset, The Cancer Genome Atlas (TCGA), we found that TCF19 is significantly increased in the lung adenocarcinoma (LAC) and squamous cell carcinoma (SCC), two primary histological subtype of NSCLC. Besides, further function cluster analysis exhibited that TCF19 may mainly participate in cell cycle. MTT, colony formation, and anchorage-independent growth ability assay confirmed that overexpression of TCF19 enhances the proliferation of both LAC and SCC cells. Besides, further experiments revealed that TCF19 contributes to cell cycle G1/S transition. Not only that, upregulation of TCF19 can inhibit the expression of p21, p27, and p57, while promote the expression of cyclin D1 by inhibiting FOXO1. Our research offers important evidence that TCF19 can promote cell-cycle progression of NSCLC cells, and TCF19 may served as novel therapeutic targets.

CUL4B regulates cancer stem-like traits of prostate cancer cells by targeting BMI1 via miR200b/c.

Cancer stem-like traits contribute to prostate cancer (PCa) progression and metastasis. Cullin 4B (CUL4B) is a member of the ubiquitin E3 ligase family and overexpressed in several solid malignancies including PCa. CUL4B has been suggested to be an oncogene through epigenetic repression of tumor suppressors. However, the link between CUL4B expression and cancer stem-like phenotype remains unclear. Western blot analysis, sphere formation, and colony formation assays were used to examine the effect of CUL4B on cancer stem-like traits in PCa cells. Mechanically, bioinformatic analysis was utilized to evaluate whether BMI1 was a target of CUL4B. Moreover, real-time polymerase chain reaction, chromatin immunoprecipitation, and luciferase reporter assays were performed to identify microRNAs regulated by CUL4B. Finally, Western blot assay was used to validate the regulation of CUL4B, miR200b, and miR200c (miR200b/c) on the stem-like characteristics of PCa cells. CUL4B promotes PCa pluripotency-associated markers expression, sphere formation, and anchorage-independent growth ability in vitro. Mechanically, CUL4B upregulates BMI1 expression via epigenetically repressing miR200b/c expression. In addition, miR200b/c could partially reverse CUL4B-induced BMI1 and pluripotency-associated marker expression. Our study revealed that CUL4B regulates cancer stem-like traits of prostate cancer cells by targeting BMI1 via miR200b/c, which might give novel insight into how CUL4B promotes PCa progression through regulating cancer stem-like traits.

Downregulation of DPF3 promotes the proliferation and motility of breast cancer cells through activating JAK2/STAT3 signaling

It is reported that the genetic variation of DPF3 is a risk factor of breast cancer through large-scale association research. However, the expression, function and mechanism in breast cancer is unknown. We applied qPCR and western blotting to detect the levels of DPF3 in breast cancer tissues. MTT and Anchorage-independent growth ability assay were used to evaluate the effect of DPF3 on cell proliferation. Wound healing and transwell invasion assay were performed to detect the role of DPF3 on cell motility ability. Herein, we found that the mRNA and protein levels of DPF3 are both significantly downregulated in breast cancer tissues. And downregulation of DPF3 can promote the proliferation and motility of breast cancer cells. Further investigation illustrated that downregulation of DPF3 can activate the JAK2/STAT3 signaling. In conclusion, we found that the downregulation of DPF3 plays an indispensable function in the progression of breast cancer, and may be served as a novel therapeutic target to therapy breast cancer.

Anti-lung Cancer and Anti-angiogenic Activities of New Designed Boronated Phenylalanine Metal Complexes.

Drug design and discovery studies still remain of great importance in the search for more convenient chemotherapeutic to avoid the drug resistance, systemic toxicity or the longterm side effects. A series of mononuclear gold (III) and platinum (II) complexes based on 4-dihydroxyboryl- DL-phenylalanine (BPA) was designed and synthesized, for the first time, by using 2, 2'-dipyridyl (L1) and 4, 4'-diaminobibenzyl (L2) ligands. Characterization of the synthesized complexes was achieved by using 1H-NMR, IR, MS and elemental analyses. MTT cell viability, endothelial tube formation, cancer cell colony formation and TRITCphalloidin cytoskeleton staining assays were performed on human umbilical vein endothelial (HUVEC) and human lung adenocarcinoma (A549) cells to establish the anticancer and anti-angiogenic activities of the complexes. It was determined that the organometallic complexes that include 2, 2'-dipyridyl ligand have higher antiproliferative activity than L2-based complexes in the micromolar range. Colony formation experiments showed that the anchorage-independent growth ability of A549s was significantly affected by the complexes in a concentration-dependent manner though L1-based complexes were more effective than L2-based ones. It was also clearly observed that the complexes have significant anti-angiogenic and cytoskeleton alterative activities. Consequently, the phenylalanine-based organometallic complexes seem to have anti-lung cancer and anti-angiogenic activities depending on the ligand type and a great potential in oncology drug development because phenylalanine amino acid has an ability to cross the cell membrane by using L-amino acid transport system. Design, synthesis and activity studies with amino acid analogs should be therefore increased to discover more efficient drugs to cure cancer diseases.

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.

Up-regulated MiR-27-3p promotes the G1-S phase transition by targeting inhibitor of growth family member 5 in osteosarcoma

ObjectiveMicroRNAs (miRNAs) play an essential role in regulating malignant progression of tumour cells by inhibiting translation or stability of messenger RNA. However, the expression pattern and regulatory mechanism of miR-27-3p in osteosarcoma remains unclear. MethodsWe examined the expression of miR-27-3p in 5 osteosarcoma cell lines compared with that in 2 normal osteocyte cell lines. Osteosarcoma cells U-2OS and MG-63 were transduced to up-regulate or down-regulate the expression of miR-27-3p. The 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide, or MTT, assay, colony formation assays, BrdUrd labelling, immunofluorescence, anchorage-independent growth ability assay and flow cytometry analysis were used to test the effect of miR-27-3p. Luciferase assays were added to verify the direct relationship between miR-27-3p and the predicted target gene inhibitor of growth family member 5 (ING5). ResultsThe expression of miR-27-3p was significantly increased in examined osteosarcoma cell lines compared with that in normal osteocyte cell lines. Up-regulation of miR-27-3p significantly accelerated osteosarcoma cell growth via promoting G1-S transition. In addition, the opposite result was observed in miR-27-3p-down-regulated cells. Up-regulation of ING5 significantly attenuated the miR-27-3p-induced proliferation in osteosarcoma cells. ConclusionsThese data suggested that miR-27-3p could promote the G1-S phase transition that leads to proliferation by down-regulating the expression of ING5 in osteosarcoma.

Nuclear EGFR-PKM2 axis induces cancer stem cell-like characteristics in irradiation-resistant cells

Radiation therapy has become an important tool in the treatment of cancer patients, but most patients relapse within 5 years. Relapse is due to the presence of cancer stem cells (CSCs), but the molecular mechanism of radioresistance in CSCs remains largely elusive. Here, we found that irradiation-resistant (IR) cells exhibited increased stem cell-like properties together with elevated anchorage-independent growth and metastasis ability. EGFR not only leads to increased acquisition of endometrial cancer stem cell markers in radioresistant sublines but is critical for the cancer stem-cell phenotype and tumorigenicity. Moreover, PKM2 functions as an interacting partner of EGFR, which induces the EMT phenotype and stem cell-like properties in IR cells. Finally, we found that the regulatory function of the EGFR-PKM2 axis is dependent on nuclear EGFR. In sum, our study indicated that EGFR and PKM2 directly interact and bind with each other to regulate the transcription of stemness-related genes and promote the stem-like phenotype, thus promoting invasion and metastasis.

Epiproteome profiling of cadmium-transformed human bronchial epithelial cells by quantitative histone post-translational modification-enzyme-linked immunosorbent assay.

Cadmium (Cd), a carcinogenic toxic metal, is pervasively distributed in the soil, water and air. Chronic exposure to Cd has been correlated to lung disease development including cancers. Although many studies have been conducted to investigate the proteome response of cells challenged with Cd, the epiproteomic responses (i.e., global histone post-translational modifications [PTMs]), particularly in human lung cells, are largely unexplored. Here, we provide an epiproteome profiling of human bronchial epithelial cells (BEAS-2B) chronically treated with cadmium chloride (CdCl2 ), with the aim of identifying global epiproteomic signatures in response to Cd epigenotoxicity. Total histone proteins from Cd-treated and untreated BEAS-2B cells were isolated and subject to quantitative histone PTM-enzyme-linked immunosorbent assay using 18 histone PTM antibodies. Our results unveiled that chronic Cd treatment led to the marked downregulation of H3K4me2 and H3K36me3 and upregulation of H3K9acS10ph, H4K5ac, H4K8ac and H4K12ac PTM marks. Cd-treated cells exhibit transformed cell properties as evidenced by enhanced cell migration and the ability of anchorage-independent growth on soft agar. Notably, treatment of Cd-transformed cells with C646, a potent histone acetyltransferase inhibitor, suppressed the expression of mesenchymal marker genes and cell migration ability of these cells. Taken together, our studies provide for the first time the global epiproteomic interrogation of chronic Cd-exposed human lung cells. The identified aberrant histone PTM alterations associated with Cd-induced epigenotoxicity likely account for the epithelial-mesenchymal transition and neoplastic survival of these cells.

Tumor Susceptibility Gene 101 Mediates Anoikis Resistance of Metastatic Thyroid Cancer Cells.

Resistance to anoikis is a pre-requisite step in metastasis, a major cause of death in patients with cancer, including thyroid cancer. Impairing anoikis resistance is a possible strategy for therapy of metastatic cancer. We, therefore, we aimed to investigate the key players of anoikis resistance. Papillary-type (BCPAP), follicular-type (FTC133), and anaplastic-type (ARO) thyroid carcinoma cells, cultured in poly(2-hydroxyethyl methacrylate)-coated plates to mimic circulating cells, were used as model systems in this study. Flow cytometry and soft-agar assays were used to determine cells exhibiting anoikis resistance. Proteomics was used to identify candidate proteins and validated using western blot and siRNA knockdown. Only ARO cells showed both anoikis resistance potential and anchorage-independent growth ability. Tumor susceptibility gene 101 protein (TSG101) was identified to be potentially important in anoikis resistance, which was confirmed by an increase in anoikis and expression of a pro-apoptotic protein (BCL-2 like protein 4) and an apoptotic marker (cleaved poly-ADP ribose polymerase) in floating siTSG101-knockdown cells. To our knowledge, this is the first study that implicates the importance of TSG101 in anoikis resistance of thyroid cancer.