Tumor Colony Formation Research Articles

Tryptophan 2,3-dioxygenase 2 controls M2 macrophages polarization to promote esophageal squamous cell carcinoma progression via AKT/GSK3β/IL-8 signaling pathway

Tryptophan 2,3-dioxygnease 2 (TDO2) is specific for metabolizing tryptophan to kynurenine (KYN), which plays a critical role in mediating immune escape of cancer. Although accumulating evidence demonstrates that TDO2 overexpression is implicated in the development and progression of multiple cancers, its tumor-promoting role in esophageal squamous cell carcinoma (ESCC) remains unclear. Here, we observed that TDO2 was overexpressed in ESCC tissues and correlated significantly with lymph node metastasis, advanced clinical stage, and unfavorable prognosis. Functional experiments showed that TDO2 promoted tumor cell proliferation, migration, and colony formation, which could be prevented by inhibition of TDO2 and aryl hydrocarbon receptor (AHR). Further experimentation demonstrated that TDO2 could promote the tumor growth of KYSE150 tumor-bearing model, tumor burden of C57BL/6 mice with ESCC induced by 4-NQO, enhance the expression of phosphorylated AKT, with subsequent phosphorylation of GSK3β, and polarization of M2 macrophages by upregulating interleukin-8 (IL-8) to accelerate tumor progression in the tumor microenvironment (TME). Collectively, our results discovered that TDO2 could upregulate IL-8 through AKT/GSK3β to direct the polarization of M2 macrophages in ESCC, and suggested that TDO2 could represent as an attractive therapeutic target and prognostic marker to ESCC.

The Discovery of a Novel Antimetastatic Bcl3 Inhibitor.

The development of antimetastatic drugs is an urgent healthcare priority for patients with cancer, because metastasis is thought to account for around 90% of cancer deaths. Current antimetastatic treatment options are limited and often associated with poor long-term survival and systemic toxicities. Bcl3, a facilitator protein of the NF-κB family, is associated with poor prognosis in a range of tumor types. Bcl3 has been directly implicated in the metastasis of tumor cells, yet is well tolerated when constitutively deleted in murine models, making it a promising therapeutic target. Here, we describe the identification and characterization of the first small-molecule Bcl3 inhibitor, by using a virtual drug design and screening approach against a computational model of the Bcl3-NF-kB1(p50) protein-protein interaction. From selected virtual screening hits, one compound (JS6) showed potent intracellular Bcl3-inhibitory activity. JS6 treatment led to reductions in Bcl3-NF-kB1 binding, tumor colony formation, and cancer cell migration in vitro; and tumor stasis and antimetastatic activity in vivo, while being devoid of overt systemic toxicity. These results represent a successful application of in silico screening in the identification of protein-protein inhibitors for novel intracellular targets, and confirm Bcl3 as a potential antimetastatic target.

Abstract PD15-09: Sox4 and smarca4 upregulate glycolysis-driven tumor proliferation through hexokinase 2 in tnbc

Abstract Background Triple-negative breast cancer (TNBC) accounts for nearly 1-in-4 breast cancer related deaths in the United States each year despite representing 10-12% of newly diagnosed cases. This aggressive disease, which is largely synonymous with the basal-like molecular subtype, predominantly affects younger women and women of African American decent. Despite a number of recent advances, overall survival has not drastically improved, highlighting the need to better understand mechanisms regulating tumor growth and progression as a means to identify novel therapeutic opportunities in these patients. Methods In order to identify the functional impact of overexpression of the oncogenic transcription factor SOX4 and its co-factor SMARCA4 in TNBC, RNAseq and ChIPseq analyses were used to identify SMARCA4-dependent and -independent SOX4 gene expression programs. Analyses of more than 3,000 tumor samples from the METABRIC and TCGA cohorts was used to investigate the functional implications and clinical association of these gene expression programs in human tumors. Mass spectrometry (MS)-based metabolomic profiling in conjunction with genetic and pharmacological-based in vitro studies were used to demonstrate the impact of these programs on tumor cell metabolism, cell viability and proliferation. Results We, and others, have previously demonstrated that the oncogenic transcription factor SOX4 is overexpressed in basal-like tumors. In order to identify mechanisms by which this protein mediates breast cancer tumorigenesis, we first identified SOX4 co-factors. Immunoprecipitation follow by MS was used to identify the SWI/SNF ATPase SMARCA4 as a prominent SOX4 cofactor that is concurrently overexpressed in basal-like tumors. Mechanistic studies showed that this complex is required to maintain an active open chromatin conformation at regulatory regions upstream of SOX4-regulated genes. ChIPseq and RNAseq-based analyses were next used to investigate the role of this complex in TNBC signaling and tumorigenesis. Our analyses identified SMARCA4-dependent and -independent SOX4 gene expression programs and demonstrated that SMARCA4-dependent SOX4 signaling is associated with poor clinical outcome. Further in silico analyses of human tumors indicate that each program mediates distinct down-stream signaling; SMARCA4-dependent signaling is associated with altered tumor metabolism and increased proliferative signaling. Consistent with this premise, in vitro studies showed that SOX4 and SMARCA4 are essential for breast tumor cell proliferation, survival and colony formation. Metabolomic profiling of breast cancer cell lines demonstrated that SOX4 overexpression resulted in increased glycolysis. The effect of SOX4 and SMARCA4 on cell metabolism was validated by demonstrating that this complex can mediate intracellular glucose consumption. Finally, we determined that SOX4 and SMARCA4 cooperatively regulate mRNA expression of hexokinase 2 (HK2), which catalyzes for the first step in glucose metabolism, and demonstrated that HK2 activity is essential for SOX4/SMARCA4-regulation of glycolysis. Importantly, in vitro genetic and pharmacological studies identified a link between HK2 activity, glycolysis and SOX4/SMARCA4-mediated cell proliferation and viability in TNBC. Conclusions Our studies have identified SOX4 and its transcriptional cofactor SMARCA4 as being uniformly overexpressed in basal-like or TNBC tumors. These analyses demonstrate that SOX4 and SMARCA4 cooperatively promote tumorigenesis in TNBC cells, in part, by modulating glycolysis and cell proliferation through transcriptional regulation of HK2. Given the drug-ability of the glycolysis pathway, these results have potential clinical implications for treatment of TNBC patients. Citation Format: Pooja Khanna, Gaurav Mehta, Michael L Gatza. Sox4 and smarca4 upregulate glycolysis-driven tumor proliferation through hexokinase 2 in tnbc [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD15-09.

CREPT/RPRD1B promotes tumorigenesis through STAT3-driven gene transcription in a p300-dependent manner

BackgroundSignal transducer and activator of transcription 3 (STAT3) has been shown to upregulate gene transcription during tumorigenesis. However, how STAT3 initiates transcription remains to be exploited. This study is to reveal the role of CREPT (cell cycle-related and elevated-expression protein in tumours, or RPRD1B) in promoting STAT3 transcriptional activity.MethodsBALB/c nude mice, CREPT overexpression or deletion cells were employed for the assay of tumour formation, chromatin immunoprecipitation, assay for transposase-accessible chromatin using sequencing.ResultsWe demonstrate that CREPT, a recently identified oncoprotein, enhances STAT3 transcriptional activity to promote tumorigenesis. CREPT expression is positively correlated with activation of STAT3 signalling in tumours. Deletion of CREPT led to a decrease, but overexpression of CREPT resulted in an increase, in STAT3-initiated tumour cell proliferation, colony formation and tumour growth. Mechanistically, CREPT interacts with phosphorylated STAT3 (p-STAT3) and facilitates p-STAT3 to recruit p300 to occupy at the promoters of STAT3-targeted genes. Therefore, CREPT and STAT3 coordinately facilitate p300-mediated acetylation of histone 3 (H3K18ac and H3K27ac), further augmenting RNA polymerase II recruitment. Accordingly, depletion of p300 abolished CREPT-enhanced STAT3 transcriptional activity.ConclusionsWe propose that CREPT is a co-activator of STAT3 for recruiting p300. Our study provides an alternative strategy for the therapy of cancers related to STAT3.

MITF functions as a tumor suppressor in non-small cell lung cancer beyond the canonically oncogenic role.

Microphthalamia-associated transcription factor (MITF) is a critical mediator in melanocyte differentiation and exerts oncogenic functions in melanoma progression. However, the role of MITF in non-small cell lung cancer (NSCLC) is still unknown. We found that MITF is dominantly expressed in the low-invasive CL1-0 lung adenocarcinoma cells and paired adjacent normal lung tissues. MITF expression is significantly associated with better overall survival and disease-free survival in NSCLC and serves as an independent prognostic marker. Silencing MITF promotes tumor cell migration, invasion and colony formation in lung adenocarcinoma cells. In xenograft mouse model, MITF knockdown enhances metastasis and tumorigenesis, but decreases angiogenesis in the Matrigel plug assay. Whole transcriptome profiling of the landscape of MITF regulation in lung adenocarcinoma indicates that MITF is involved in cell development, cell cycle, inflammation and WNT signaling pathways. Chromatin immunoprecipitation assays revealed that MITF targets the promoters of FZD7, PTGR1 and ANXA1. Moreover, silencing FZD7 reduces the invasiveness that is promoted by silencing MITF. Strikingly, MITF has significantly inverse correlations with the expression of its downstream genes in lung adenocarcinoma. In summary, we demonstrate the suppressive role of MITF in lung cancer progression, which is opposite to the canonical oncogenic function of MITF in melanoma.

Abstract PO-035: SOX4 and SMARCA4 upregulate glycolysis-driven tumor proliferation through Hexokinase 2 in Triple Negative Breast Cancer

Abstract SOX4 is a well-established oncogenic transcription factor and has been shown to be associated with malignant transformation and metastasis in several cancer types including breast, prostate, acute lymphoblastic leukemia, and melanoma. While SOX4 is overexpressed in ~85% of TNBCs (Triple Negative Breast Cancers), the mechanisms by which it contributes to activation of pro-proliferative or pro-survival signaling in these tumors remains unclear. We determined that SOX4 forms a novel and essential complex with the SWI/SNF ATPase SMARCA4 and demonstrated that this complex is required to maintain an open and active chromatin conformation at the promoter and enhancer regions of SOX4-regulated genes. An integrated analysis of ChIPseq and RNAseq data was used to identify SMARCA4-dependent and SMARCA4-independent SOX4 gene expression programs and enrichment analyses indicated that these programs are responsible for unique down-stream signaling networks. SMARCA4-dependent SOX4 signaling was found to be correlate with a poor prognosis in TNBC tumors and was associated with increased cell proliferation and activation of pro-oncogenic processes, including glycolysis. Consistent with these data, in vitro studies showed that SOX4 and SMARCA4 are essential for TNBC breast tumor cell proliferation, survival and colony formation. Mass spectrometry-based metabolomic profiling was used to demonstrate that SOX4 can induce glycolysis as evident by increased glucose-6-phosphate, glyceralehyde-3-phosphate, dihydroxyacetone phosphate, 2-phosphoglycerate, pyruvate and lactate following SOX4 overexpression and the effect of SOX4 and SMARCA4 on glucose consumption was confirmed in vitro. Mechanistically, we determined that SOX4 and SMARCA4 cooperate to transcriptionally regulate expression of Hexokinase 2 (HK2), which catalyzes the first step in glucose metabolism, and that increased expression of HK2 is required to mediate SOX4-dependent glycolysis. Finally we demonstrate that genetic or pharmacological inhibition of HK2 results in reduced TNBC cell proliferation and growth. Collectively, our data suggest that SOX4 and SMARCA4 cooperate to regulate glycolysis and cell proliferation in TNBC in part through activation of HK2 expression. Given the noted association between SOX4 expression and increased tumor proliferation and aggressiveness in multiple forms of cancer, as well as evidence that SOX4 can regulate SMARCA4 expression, these data suggest that aberrant SOX4 activity may have a similar effect on metabolic and cell signaling across multiple tumor types. Citation Format: Pooja Khanna, Gaurav Mehta, Vrushank Bhatt, Jessie Y. Guo, Michael L. Gatza. SOX4 and SMARCA4 upregulate glycolysis-driven tumor proliferation through Hexokinase 2 in Triple Negative Breast Cancer [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-035.

CTEN Induces Tumour Cell Invasion and Survival and Is Prognostic in Radiotherapy-Treated Head and Neck Cancer.

Simple SummaryC-terminal tensin-like, or CTEN, is a cytoskeletal protein that is expressed highly in head and neck cancer (HNSCC). We studied CTEN function using gene knockdown and found that CTEN contributes to HNSCC progression in several ways, promoting tumour cell invasion and also cell survival. Notably, CTEN expression protects tumour cells from radiation-induced apoptosis and consistent with this, we found that CTEN expression predicts for survival in patients treated with radiotherapy (but not surgery), suggesting that CTEN may have utility as a predictive marker of radiotherapy resistance.Head and neck squamous cell carcinoma (HNSCC) is a heterogenous disease treated with surgery and/or (chemo) radiotherapy, but up to 50% of patients with late-stage disease develop locoregional recurrence. Determining the mechanisms underpinning treatment resistance could identify new therapeutic targets and aid treatment selection. C-terminal tensin-like (CTEN) is a member of the tensin family, upregulated in several cancers, although its expression and function in HNSCC are unknown. We found that CTEN is commonly upregulated in HNSCC, particularly HPV−ve tumours. In vitro CTEN was upregulated in HPV−ve (n = 5) and HPV+ve (n = 2) HNSCC cell lines. Stable shRNA knockdown of CTEN in vivo significantly reduced tumour growth (SCC-25), and functional analyses in vitro showed that CTEN promoted tumour cell invasion, colony formation and growth in 3D-culture (SCC-25, Detroit 562). RNA sequencing of SCC-25 cells following CTEN siRNA knockdown identified 349 differentially expressed genes (logFC > 1, p < 0.05). Gene ontology analysis highlighted terms relating to cell locomotion and apoptosis, consistent with in vitro findings. A membrane-based antibody array confirmed that CTEN regulated multiple apoptosis-associated proteins, including HSP60 and cleaved caspase-3. Notably, in a mixed cohort of HPV+ve and HPV−ve HNSCC patients (n = 259), we found a significant, independent negative association of CTEN with prognosis, limited to those patients treated with (chemo)radiotherapy, not surgery, irrespective of human papillomavirus (HPV) status. These data show that CTEN is commonly upregulated in HNSCC and exerts several functional effects. Its potential role in modulating apoptotic response to therapy suggests utility as a predictive biomarker or radio-sensitising target.

FANCD2 Confers a Malignant Phenotype in Esophageal Squamous Cell Carcinoma by Regulating Cell Cycle Progression.

Simple SummaryFanconi anemia patients with germline FANCD2 defects are susceptible to cancers. In order to understand FANCD2 function in esophageal cancers, we used cell line, animal model, and sequencing approaches. We knocked out the FANCD2 gene and examined the functional impact of its loss on tumor growth and metastasis and performed assays for cell growth, cell cycle, and cellular localization. FANCD2 promotes tumorigenesis in this cancer. FANCD2 is significantly upregulated in tumors. Depletion of FANCD2 protein expression significantly suppresses the cancer cell proliferation and tumor colony formation and metastasis potential, as well as cell cycle progression, by involving specific cell signaling pathways. FANCD2 is moved out of the nucleus to the cytoplasm during cell cycle progression. We provide evidence of a novel role of FANCD2 in esophageal cancer progression and its potential value as a biomarker for disease management.Fanconi anemia patients with germline genetic defects in FANCD2 are highly susceptible to cancers. Esophageal squamous cell carcinoma (ESCC) is a deadly cancer. Little is known about the function of FANCD2 in ESCC. For detailed molecular and mechanistic insights on the functional role of FANCD2 in ESCC, in vivo and in vitro assays and RNA sequencing approaches were used. Utilizing Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) technology, FANCD2 knockout models were established to examine the functional impact in mouse models for tumor growth and metastasis and in vitro assays for cell growth, cell cycle, and cellular localization. Our RNA sequence analyses were integrated with public datasets. FANCD2 confers a malignant phenotype in ESCC. FANCD2 is significantly upregulated in ESCC tumors, as compared to normal counterparts. Depletion of FANCD2 protein expression significantly suppresses the cancer cell proliferation and tumor colony formation and metastasis potential, as well as cell cycle progression, by involving cyclin-CDK and ATR/ATM signaling. FANCD2 translocates from the nucleus to the cytoplasm during cell cycle progression. We provide evidence of a novel role of FANCD2 in ESCC tumor progression and its potential usefulness as a biomarker for ESCC disease management.

A Novel Therapeutic siRNA Nanoparticle Designed for Dual-Targeting CD44 and Gli1 of Gastric Cancer Stem Cells.

PurposeGastric cancer stem cells (CSCs) are important for the initiation, growth, recurrence, and metastasis of gastric cancer, due to their chemo-resistance and indefinite proliferation. Herein, to eliminate gastric CSCs, we developed novel CSC-targeting glioma-associated oncogene homolog 1 (Gli1) small interfering RNA (siRNA) nanoparticles that are specifically guided by a di-stearoyl-phosphatidyl-ethanolamine- hyaluronic-acid (DSPE-HA) single-point conjugate, as an intrinsic ligand of the CD44 receptor. We refer to these as targeting Gli1 siRNA nanoparticles.MethodsWe used the reductive amination reaction method for attaching amine groups of DSPE to aldehydic group of hyaluronic acid (HA) at the reducing end, to synthesize the DSPE-HA single-point conjugate. Next, targeting Gli1 siRNA nanoparticles were prepared using the layer-by-layer assembly method. We characterized the stem cellular features of targeting Gli1 siRNA nanoparticles, including their targeting efficiency, self-renewal capacity, the migration and invasion capacity of gastric CSCs, and the penetration ability of 3D tumor spheroids. Next, we evaluated the therapeutic efficacy of the targeting Gli1 siRNA nanoparticles by using in vivo relapsed tumor models of gastric CSCs.ResultsCompared with the multipoint conjugates, DSPE-HA single-point conjugates on the surface of nanoparticles showed significantly higher binding affinities with CD44. The targeting Gli1 siRNA nanoparticles significantly decreased Gli1 protein expression, inhibited CSC tumor spheroid and colony formation, and suppressed cell migration and invasion. Furthermore, in vivo imaging demonstrated that targeting Gli1 siRNA nanoparticles accumulated in tumor tissues, showing significant antitumor recurrence efficacy in vivo.ConclusionIn summary, our targeting Gli1 siRNA nanoparticles significantly inhibited CSC malignancy features by specifically blocking Hedgehog (Hh) signaling both in vitro and in vivo, suggesting that this novel siRNA delivery system that specifically eliminates gastric CSCs provides a promising targeted therapeutic strategy for gastric cancer treatment.

Abstract 5039: SCD1/FADS2 and ferroptosis signalings orchestrate ovarian cancer cell metabolism and metastatic progression in peritoneal metastases

Abstract Peritoneal metastases are common and usually associated with poor prognosis in advanced ovarian cancers. Emerging evidence indicates that the ascites microenvironment provides a key reservoir of growth factors and free fatty acids for promoting metastatic recurrence and chemoresistance of ovarian cancers. However, how the metastatic ovarian cancer cells adapt and utilize such a unique tumor microenvironment remains elusive. Here, we report that ovarian cancer cells co-cultured in ascites or omental conditioned medium (OCM) enhance their oncogenic capacities through elevated lipid metabolism. RNA sequencing (RNA-seq) and lipidomic profiling analyses revealed two key mediators of UFAs, Stearoyl-CoA Desaturase-1 (SCD1) and Fatty acid Desaturase-2 (FADS2) were upregulated accompanied with the accumulation of poly- and mono-unsaturated fatty acids (UFAs) in ovarian cancer cells co-cultured in OCM. Enforced expression of either SCD1 or FADS2 promotes cancer stemness, in vitro and ex vivo tumor colony formation, and cell migration/invasion through activation of NFκB signaling. SCD1/FADS2 increases not only UFAs accumulation but also the cell membrane fluidity which is crucial for the epithelial-mesenchymal transition (EMT) and metastatic potentials. On the other hand, RNA-Seq analysis on SCD1/FADS2-depleted ovarian cancer cells showed the ferroptotic activities were inversely correlated with SCD1/FADS2. Of note, either knockout of SCD1/FADS2 or co-treatment of Erastin could enhance cisplatin-induced cytotoxicity through the elevation of iron-mediated reactive oxygen species (ROS), and subsequently led ferroptosis by upregulation of GPX4/xCT expressions in ovarian cancer cells. Higher tumorigenic capacity expressed in iron-rich ovarian cancer cells, which contains sphere formation, EMT potential and cell viability. Taken together, SCD1/FADS2 and ferroptosis signaling pathways orchestrate the metabolic activities, ROS stresses and cellular iron homeostasis during the metastatic progression of ovarian cancer cells in peritoneum microenvironment. Hence, targeting these signaling cascades by the drug cocktail/combination strategy could reduce the risk of metastatic recurrence and chemoresistance in advanced ovarian cancers. Citation Format: Yang Xuan, Mingo MH Yung, Arvin FS Chen, Hextan YS Ngan, David W Chan. SCD1/FADS2 and ferroptosis signalings orchestrate ovarian cancer cell metabolism and metastatic progression in peritoneal metastases [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5039.

In vitro antitumor and anti-angiogenic activities of a shrimp chondroitin sulfate

Thrombin triggers cellular responses that are crucial for development and progression of cancer, such as proliferation, migration, oncogene expression and angiogenesis. Thus, biomolecules capable of inhibiting this protease have become targets in cancer research. The present work describes the in vitro antitumor properties of a chondroitin sulfate with anti-thrombin activity, isolated from the Litopenaeus vannamei shrimp (sCS). Although the compound was unable to induce cytotoxicity or cell death and/or cell cycle changes after 24 h incubation, it showed a long-term antiproliferative effect, reducing the tumor colony formation of melanoma cells by 75% at 100 μg/mL concentration and inhibiting the anchorage-independent colony formation. sCS reduced 66% of melanoma cell migration in the wound healing assay and 70% in the transwell assay. The compound also decreased melanin and TNF-α content of melanoma cells by 52% and 75% respectively. Anti-angiogenic experiments showed that sCS promoted 100% reduction of tubular structure formation at 100 μg/mL. These results are in accordance with the sCS-mediated in vitro expression of genes related to melanoma development (Cx-43, MAPK, RhoA, PAFR, NFKB1 and VEGFA). These findings bring a new insight to CS molecules in cancer biology that can contribute to ongoing studies for new approaches in designing anti-tumor therapy.

Safety of menstrual blood-derived stromal cell transplantation in treatment of intrauterine adhesion.

Intrauterine adhesion (IUA) can cause serious damage to women's reproductive health, yet current treatment methods are difficult to achieve satisfactory results. In our previous studies, we demonstrated that menstrual-derived stromal stem cells (MenSCs), with high proliferative capacity and self-renewal ability, have a powerful therapeutic effect in patients with severe IUA. However, safety assessment of MenSCs transplantation is essential for its further application. To evaluate the short-, medium-, and long-term biosafety of MenSCs via intrauterine transplantation in a rat model of IUA, with a focus on toxicity and tumorigenicity. MenSCs were injected into the sub-serosal layer of the uterus in an IUA rat model, for 3 d, 3 mo, and 6 mo separately, to monitor the corresponding acute, sub-chronic, and chronic effects. Healthy rats of the same age served as negative controls. Toxicity effects were evaluated by body weight, organ weight, histopathology, hematology, and biochemistry tests. Tumorigenicity of MenSCs was investigated in Balb/c-nu mice in vivo and by colony formation assays in vitro. Compared with the same week-old control group, all of the IUA rats receiving MenSC transplantation demonstrated no obvious changes in body weight, main organ weight, or blood cell composition during the acute, sub-chronic, and chronic observation periods. At the same time, serum biochemical tests showed no adverse effects on metabolism or liver and kidney function. After 4 wk of subcutaneous injection of MenSCs in Balb/c-nu nude mice, no tumor formation or cell metastasis was observed. Moreover, there was no tumor colony formation of MenSCs during soft agar culture in vitro. There is no acute, sub-chronic, or chronic poisoning, infection, tumorigenesis, or endometriosis in rats with IUA after MenSC transplantation. The above results suggest that intrauterine transplantation of MenSCs is safe for endometrial treatment.

LncRNA AFAP1-AS1 promotes triple negative breast cancer cell proliferation and invasion via targeting miR-145 to regulate MTH1 expression

The actin fiber-associated protein 1-antisense RNA1 (AFAP1-AS1) is upregulated in various cancers and associated with cancer proliferation and metastasis. Several cancer-related pathways have been linked to up-expression of this long non-coding (lnc)RNA, but the underlying mechanisms are yet unknown. In triple negative breast cancer (TNBC), AFAP1-AS1 expression is also significantly overexpressed compared to that in other subtypes of breast cancer from the TCGA dataset. In this study, we performed bioinformatic RNAhybrid analyses and identified that miR-145 is a potential target of AFAP1-AS1 and able to reduce MutT homolog-1 (MTH1) expression. Thus, this study investigated the oncogenic activity of AFAP1-AS1 in TNBC cells and the underlying mechanisms that are yet poorly understood. The results showed that miR-145 expression was low, whereas AFAP1-AS1 and MTH1 expression was high in TNBC cells and that miR-145 mimics reduced TNBC cell proliferation and invasion, whereas miR-145 knockdown exerted the opposite activity in TNBC cells. Moreover, knockdown of AFAP1-AS1 reduced tumor cell proliferation and invasion, but miR-145 co-transfection rescued tumor cell viability and colony formation ability. The dual luciferase reporter assay showed that AFAP1-AS1 could directly target miR-145, while miR-145 could directly target MTH1. After knockdown of ATF6, AFAP1-AS1 was reduced along with AFAP1-AS1 promoter activity. This study revealed that AFAP1-AS1 could promote TNBC cell proliferation and invasion via regulation of MTH1 expression through targeting of miR-145.

Downregulation of SFRP1 is a protumorigenic event in hepatoblastoma and correlates with beta-catenin mutations

BackgroundHepatoblastoma (HB) and pediatric hepatocellular carcinoma (HCC) are the most common malignant liver tumors in childhood. Both tumor types exhibit genetic and epigenetic alterations in the WNT/β-catenin signaling pathway, which is a key regulator of liver progenitor cells in embryonic development. The tumors demonstrate a high rate of β-catenin mutations and gene expression changes of several WNT antagonists. However, the role of the WNT inhibitory factor secreted frizzled-related protein 1 (SFRP1) has not been addressed in pediatric liver cancer so far.ResultsIn our study, we investigated the gene expression level, DNA methylation status and functional relevance of SFRP1 in HB cell lines and in pediatric liver tumor patient samples. SFRP1 was downregulated due to DNA promoter methylation in all tested HB cell lines. Overexpression of SFRP1 in HB cell lines diminished tumor cell proliferation, colony formation and migration potential. In addition, the SFRP1-expressing HB cell lines showed reduced WNT/β-catenin signaling pathway activity and decreased expression of WNT target genes. To evaluate the utility of SFRP1 as a biomarker in pediatric liver cancer, we determined the gene expression level and DNA methylation status of SFRP1 in 45 pediatric liver tumor patient samples. The correlation analysis of different clinical parameters and tumor characteristics revealed a significant correlation of reduced SFRP1 expression with the presence of mutant β-catenin. The methylation status of SFRP1 was furthermore associated to a pediatric liver tumor type with HCC-like characteristics, TERT mutations and an older age at diagnosis.ConclusionAltogether, our data demonstrate that the epigenetic suppression of the WNT/β-catenin antagonist SFRP1 has an important impact on the malignant behavior of HB cells. Although SFRP1 methylation is a common event in HCC-like pediatric liver tumors, its potential as a prognostic or diagnostic biomarker needs to be further investigated.

Tumor cell-intrinsic PD-1 receptor is a tumor suppressor and mediates resistance to PD-1 blockade therapy

The programmed cell death 1 (PD-1) receptor on the surface of immune cells is an immune checkpoint molecule that mediates the immune escape of tumor cells. Consequently, antibodies targeting PD-1 have shown efficacy in enhancing the antitumor activity of T cells in some types of cancers. However, the potential effects of PD-1 on tumor cells remain largely unknown. Here, we show that PD-1 is expressed across a broad range of tumor cells. The silencing of PD-1 or its ligand, PD-1 ligand 1 (PD-L1), promotes cell proliferation and colony formation in vitro and tumor growth in vivo. Conversely, overexpression of PD-1 or PD-L1 inhibits tumor cell proliferation and colony formation. Moreover, blocking antibodies targeting PD-1 or PD-L1 promote tumor growth in cell cultures and xenografts. Mechanistically, the coordination of PD-1 and PD-L1 activates its major downstream signaling pathways including the AKT and ERK1/2 pathways, thus enhancing tumor cell growth. This study demonstrates that PD-1/PD-L1 is a potential tumor suppressor and potentially regulates the response to anti-PD-1/PD-L1 treatments, thus representing a potential biomarker for the optimal cancer immunotherapeutic treatment.

RETRACTED: Silencing of p68 and STAT3 synergistically diminishes cancer progression

AimsSince several factors are involved in the tumorigenesis process, targeting only one factor most probably cannot overwhelm cancer progression. Therefore, it seems that combination therapy through targeting more than one cancer-related factor may lead to cancer control. The expression and function of p68 (DDX5; DEAD-Box Helicase 5) are dysregulated in various cancers. P68 is also a co-activator of many oncogenic transcription factors such as the signal transducer and activator of transcription-3 (STAT3), which contributes to cancer progression. This close connection between p68 and STAT3 plays an important role in the growth and development of cancer. Materials and methodsWe decided to suppress the p68/STAT3 axis in various cancer cells by using Polyethylene glycol-trimethyl Chitosan-Hyaluronic acid (PEG-TMC-HA) nanoparticles (NPs) loaded with siRNA molecules. We assessed the impact of this combination therapy on apoptosis, proliferation, angiogenesis, and tumor growth, both in vitro and in vivo. Key findingsThe results showed that siRNA-loaded NPs notably suppressed the expression of p68/STAT3 axis in cancer cells, which was associated with blockade of tumor growth, colony formation, angiogenesis, and cancer cell migration. In addition to apoptosis induction, this combined therapy also reduced the expression of several tumor-promoting factors including Fibroblast growth factors (FGF), vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), matrix metallopeptidases-2 (MMP-2), MMP-9, hypoxia-inducible factor-(HIF-1α), interleukin-6 (IL-6), IL-33, Bcl-x, vimentin, and snail. SignificanceThese findings indicate the potential of this nano-based anti-cancer therapeutic strategy for efficient cancer therapy which should be further investigated in future studies.

MicroRNA-144 inhibits cell proliferation and invasion by directly targeting TIGAR in esophageal carcinoma.

microRNAs (miRNAs) have been identified to play vital roles in the development and progression of numerous different types of human malignancy, including esophageal squamous cell carcinoma (ESCC). In the present study, the biological function of microRNA-144 (miR-144) was investigated, as well as its underlying molecular mechanism in ESCC. The results revealed that miR-144 expression was significantly decreased, whereas the expression of TP53-inducible glycolysis and apoptosis regulator (TIGAR) was significantly increased in human ESCC tissues when compared with adjacent non-tumor tissues. An increase in TIGAR was significantly associated with tumor size and Tumor-Node-Metastasis staging in patients. Functional analysis revealed that the overexpression of miR-144 using lentivirus particles significantly inhibited cell proliferation and tumor colony formation, and induced cell apoptosis in EC9706 and EC109 cells. The autophagy activity was also enhanced by miR-144 activity. In addition, overexpression of miR-144 significantly inhibited tumor growth in vivo. In the present study, TIGAR was confirmed to be the downstream target of miR-144 in ESCC. siRNA-mediated downregulation of TIGAR inversely regulated the inhibition effect of miR-144 on ESCC cells. To conclude, the present study demonstrated that miR-144 inhibits proliferation and invasion in esophageal cancer by directly targeting TIGAR.

Inhibition of Long Noncoding RNA CRNDE Increases Chemosensitivity of Medulloblastoma Cells by Targeting miR-29c-3p.

Long noncoding RNA CRNDE (CRNDE) recently emerged as a carcinogenic promoter in various cancers including medulloblastoma. However, the functions and molecular mechanisms of CRNDE to the acquired drug resistance of medulloblastoma are still unclear. The transcript levels of CRNDE were examined in four medulloblastoma cell lines exposed to cisplatin treatment, and IC50 values were calculated. Effects of CRNDE knockdown or miR-29c-3p overexpression on cell viability, colony formation, apoptosis, migration, and invasion were assessed using the CCK-8, colony formation assay, flow cytometry, and Transwell assays, respectively. RNA pulldown and RNA-binding protein immunoprecipitation (RIP) were performed to confirm the molecular interactions between CRNDE and miR-29c-3p involved in medulloblastoma cells. The in vivo role of CRNDE knockdown or miR-29c-3p overexpression on tumor growth and apoptosis was evaluated in a xenograft mouse model of human medulloblastoma. The transcript levels of lncRNA CRNDE were significantly higher in cisplatin-treated tumor cells with higher IC50 values. Depletion of CRNDE inhibited tumor cell proliferation and colony formation, induced cell apoptosis, and suppressed migration and invasion in medulloblastoma cells. Moreover, overexpression of miR-29c-3p inhibited tumor cell proliferation and colony formation, migration, and invasion, and enhanced apoptosis and chemosensitivity to cisplatin. In addition, CRNDE was found to act as a miR-29c-3p sponge. Furthermore, in vivo experiments showed the CRNDE/miR-29c-3p interactions involved in medulloblastoma. Our study demonstrates that CRNDE acts as a critical mediator of proliferation, apoptosis, migration, invasion, and resistance to chemotherapeutics via binding to and negatively regulating miR-29c-3p in medulloblastoma cells. These results provide novel molecular targets for treatment of medulloblastoma.

SET domain-containing 5 is a potential prognostic biomarker that promotes esophageal squamous cell carcinoma stemness

SET domain-containing 5 (SETD5) is an uncharacterized member of the protein lysine methyltransferase family. Although it was reported that SETD5 gene mutations are associated with the several types of human cancer, its functional role in esophageal squamous cell carcinoma (ESCC) progression has not been fully elucidated. In the present study, we used tissue samples from 147 patients with ESCC and ESCC cell lines to determine the clinicopathological significance of SETD5 in ESCC and its effects on ESCC stemness. We performed immunohistochemical staining, immunofluorescence imaging, and tumor sphere formation, colony formation, flow cytometry, wound healing, Transwell, and western blotting assays. SETD5 expression was upregulated in ESCC tissue and associated with primary tumor (pT) stage, clinical stage, lymph node metastasis, shorter overall survival rate, and disease-free survival rate. Cox regression analyses indicated that SETD5 is an independent poor prognostic factor of ESCC. In addition, SETD5 expression was correlated with cancer stemness-related protein, hypoxia-inducible factor-1α (HIF-1α), and CD68 expression. Moreover, immunofluorescence analysis revealed that SETD5 was co-localized with CD44 and SOX2 in TE10 and TE11 cells and that exposing cells to cobalt chloride increased HIF-1α, SETD5, and stemness-related protein expression in a time-dependent manner. Furthermore, SETD5 expression was significantly correlated with the expression of cell cycle-related genes and PI3K/Akt signaling pathway-related proteins. Finally, knocking down SETD5 downregulated the expression of stemness-related and PI3K/Akt signaling pathway proteins, while inhibiting tumor spheroid formation, cell proliferation, migration, and invasion in ESCC cells. These results indicate that SETD5 expression is associated with cancer stemness and that SETD5 is a potential prognostic biomarker and therapeutic target for ESCC.

A comprehensive in vivo and mathematic modeling-based kinetic characterization for aspirin-induced chemoprevention in colorectal cancer.

Accumulating evidence suggests that aspirin has anti-tumorigenic properties in colorectal cancer (CRC). Herein, we undertook a comprehensive and systematic series of in vivo animal experiments followed by 3D-mathematical modeling to determine the kinetics of aspirin's anti-cancer effects on CRC growth. In this study, CRC xenografts were generated using four CRC cell lines with and without PIK3CA mutations and microsatellite instability, and the animals were administered with various aspirin doses (0, 15, 50, and 100 mg/kg) for 2 weeks. Cell proliferation, apoptosis and protein expression were evaluated, followed by 3D-mathematical modeling analysis to estimate cellular division and death rates and their impact on aspirin-mediated changes on tumor growth. We observed that aspirin resulted in a dose-dependent decrease in the cell division rate, and a concomitant increase in the cell death rates in xenografts from all cell lines. Aspirin significantly inhibited cell proliferation as measured by Ki67 staining (P < 0.05-0.01). The negative effect of aspirin on the rate of tumor cell proliferation was more significant in xenograft tumors derived from PIK3CA mutant versus wild-type cells. A computational model of 3D-tumor growth suggests that the growth inhibitory effect of aspirin on the tumor growth kinetics is due to a reduction of tumor colony formation, and that this effect is sufficiently strong to be an important contributor to the reduction of CRC incidence in aspirin-treated patients. In conclusion, we provide a detailed kinetics of aspirin-mediated inhibition of tumor cell proliferation, which support the epidemiological data for the observed protective effect of aspirin in CRC patients.