Soft Agar Colony Formation Assay Research Articles

Ruxolitinib-loaded poly-ɛ-caprolactone (PCL) nanoparticles inhibit JAK2/STAT5 signaling in BT474 breast cancer cells by downregulating Bcl-2 and Mcl-1.

JAK/STAT signaling plays an important role in regulating cell proliferation. Reducing proliferation and inducing cell death with gene-specific inhibitors such as ruxolitinib, Receptor tyrosine kinases (RTK) inhibitor targeting JAK1/2, are therapeutic approaches. The use of nanoparticles can reduce the toxicity and side effects of drugs, as they act directly on cancer cells and can selectively increase drug accumulation in tumor cells. Poly-ɛ-caprolactone (PCL) is a polymer that is frequently used in drug development. In this study, Rux-PCL-NPs were synthesized to increase the effectiveness of ruxolitinib. In addition, this study aimed to determine the effect of Rux-PCL-NPs on JAK/STAT signaling and apoptotic cell death. Rux-PCL-NPs were synthesized by nanoprecipitation. The Rux-PCL-NPs had a spherical and mean particle size of 219 ± 88.66nm and a zeta potential of 0.471 ± 0.453 mV. In vitro cytotoxicity and antiproliferative effects were determined by MTT and soft agar colony formation assays, respectively. The effects of ruxolitinib, PCL-NPs, and Rux-PCL-NPs on apoptosis and the JAK/STAT pathway in cells were examined by western blot analysis. PCL-NPs did not have a toxic effect on the cells. The IC50 value of Rux-PCL-NPs was decreased 50-fold compared to that of ruxolitinib. Rux-PCL-NPs promoted cell death by downregulating JAK2 and STAT5, thereby inhibiting the JAK/STAT pathway. Our results revealed that Rux-PCL-NPs, which increased the efficacy of ruxolitinib, regulated apoptosis and the JAK2/STAT5 pathway.

Silencing LINC00987 ameliorates adriamycin resistance of acute myeloid leukemia via miR-4458/HMGA2 axis

BackgroundMost patients with acute myeloid leukemia (AML) eventually develop drug resistance, leading to a poor prognosis. Dysregulated long gene non coding RNAs (lincRNAs) have been implicated in chemoresistance in AML. Unfortunately, the effects of lincRNAs which participate in regulating the Adriamycin (ADR) resistance in AML cells remain unclear. Thus, the purpose of this study is to determine LINC00987 function in ADR-resistant AML.MethodsIn this study, ADR-resistant cells were constructed. LINC00987, miRNAs, and HMGA2 mRNA expression were measured by qRT-PCR. P-GP, BCRP, and HMGA2 protein were measured by Western blot. The proliferation was analyzed by MTS and calculated IC50. Soft agar colony formation assay and TUNEL staining were used to analyze cell colony formation and apoptosis. Xenograft tumor experiment was used to analyze the xenograft tumor growth of ADR-resistant AML.ResultsWe found that higher expression of LINC00987 was observed in AML patients and associated with poor overall survival in AML patients. LINC00987 expression was increased in ADR-resistant AML cells, including ADR/MOLM13 and ADR/HL-60 cells. LINC00987 downregulation reduces ADR resistance in ADR/MOLM13 and ADR/HL-60 cells in vitro and in vivo, while LINC00987 overexpression enhanced ADR resistance in MOLM13 and HL-60 cells. Additionally, LINC00987 functions as a competing endogenous RNA for miR-4458 to affect ADR resistance in ADR/MOLM13 and ADR/HL-60 cells. HMGA2 is a target of miR-4458. LINC00987 knockdown and miR-4458 overexpression reduced HMGA2 expression. HMGA2 overexpression enhanced ADR resistance, which reversed the function of LINC00987 silencing in suppressing ADR resistance of ADR/MOLM13 and ADR/HL-60 cells.ConclusionsDownregulation of LINC00987 weakens ADR resistance by releasing miR-4458 to deplete HMGA2 in ADR/MOLM13 and ADR/HL-60. Therefore, LINC00987 may act as the therapeutic target for treating chemoresistant AML.

Knock out of ELAVL1 affects steroid synthesis in adrenocortical carcinoma cell line NCI-H295R and leads to a less malignant phenotype

Introduction: Adrenocortical carcinoma (ACC) is a rare malignancy of the adrenal gland. With a 5-year-survival rate below 50% and no curative treatment options in advanced stages new therapeutic options in ACC are needed. Analyzing the ACC cohort of the cancer genome atlas we found a significant negative correlation of the expression level of a gene called Embryonic Lethal Abnormal Vision Like 1 (ELAVL1) and ACC patient survival. ELAVL1 has been described to be unfavorable in a lot of different tumor entities, but its function in ACC progression is still unknown. Methods: To evaluate the function of ELAVL1 in ACC progression we performed a CRISPR/Cas9 mediated knock out (KO) of the ELAVL1 gene in the ACC cell line NCI-H295R. The KO was confirmed on DNA level via Sanger sequencing and on protein level via immunofluorescence and western blot. Transcriptome changes were analyzed by next generation RNA sequencing (NGS). To characterize changes in cell function we performed soft agar colony forming assays and evaluated changes in steroid production via mass spectrometry. Results: Via CRISPR/Cas9 we knocked out ELAVL1 in NCI-H295R cells. NGS data showed 6926 significantly deregulated genes in ELAVL1-KO cells compared to control cells. There was an upregulation of 3468 genes, while 3458 genes showed a lower expression in ELAVL1 KO cells. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed a significant upregulation of 29 genes associated with aldosterone synthesis and a significant downregulation of 87 genes associated with pathways in cancer. Analyzing cell function via soft agar colony forming assays an impaired colony forming ability in ELAVL1 KO cells was seen. Furthermore, we could prove significant changes in steroid production. ELAVL1 KO led to an inhibition of androgen and glucocorticoid synthesis and an enhancement in mineralocorticoid synthesis. These changes could not only be proven by measuring steroid concentrations in cell culture supernatants, but also by NGS showing a downregulation of the genes CYP17A1 and CYP11B1 and an upregulation of CYP21A2 and CYP11B2. Conclusion: Our study showed that ELAVL1 affects the regulation of around 7000 genes in the ACC cell line NCI-H295R. Especially the inhibition of glucocorticoid synthesis shows that ELAVL1 may be a new target for therapeutic approaches in ACC treatment since hypercortisolism is one of the main symptoms in ACC and a negative prognostic factor for patient survival. The authors have nothing to disclose. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 4760: Inhibition of FASN postpones development of resistance to BRAF inhibitors in colorectal cancer

Abstract Introduction: Mutations in proto-oncogene BRAF occur in about 10-15% of CRC patients and BRAFV600E is the most common. For patients who undergo BRAF-targeted therapy, resistance develops 4-6 months after treatment initiation and results in more aggressive disease. We found that development of resistance to BRAF inhibitors (BRAFi) is associated with an increase in lipid metabolism and expression of fatty acid synthase (FASN). FASN is a key enzyme of lipid synthesis overexpressed in CRC. Therefore, our hypothesis is that inhibition lipid metabolism via FASN will postpone development of resistance to BRAFi. Methods: We established CRC cells resistant to PLX8394, a novel BRAFi. To evaluate differences in parental and resistance cells, CellTiterGlo 2.0, PrestoBlue, CytoSelectTM Invasion Assay, Triglyceride Assay, Seahorse XF, and confocal microscopy were used. Combination of PLX8394 and TVB3664 or C75 (FASN inhibitors) was tested on cell viability, colony formation, and synergy studies in parental and BRAFi resistant cells. Results: The development of resistance to BRAFi promotes cellular proliferation and increases cyclin D and survivin expression in vitro and in vivo. BRAFi resistance is also associated with an increase in invasive properties and loss of E-cadherin expression. Metabolic changes include an increase in lipid metabolism, oxidative phosphorylation, and triglycerides storage. RNAseq and western blot analysis show significant upregulation of FASN in BRAFi resistant cells. Using cell viability and soft agar colony formation assays, we show that combined PLX8394 and TVB3664 treatment leads to a significantly higher decrease in cell viability and colony formation as compared to each drug alone in parental cells but not in BRAFi resistant cells. The calculation of a Bliss synergy score confirms that combination treatment with C75 and PLX8394 has synergetic effect in BRAFV600E cells. To further confirm that FASN contributes to resistance to BRAFi, we show that HT29 FASN shRNA cells are more susceptible to PLX8394 treatment as compared to control cells. Importantly, our data show that the long-term treatment with PLX8394 in combination with TVB3664 postpones development of resistance to BRAFi as compared to cells treated with PLX8394 alone. Conclusion: Our study demonstrates that resistance to BRAFi is associated with a significant increase in proliferation, metastasis, and lipid metabolism. We demonstrate that combination of FASN inhibitors and BRAFi postpones development of resistance in BRAFV600E cells. However, FASN inhibition does not sensitize cells to BRAFi in already resistance cells, suggesting that this approach cannot be used to overcome acquired resistance to BRAFi. In summary our data suggest that an addition of TVB3664 at the beginning of BRAF treatment regimen could be an efficacious treatment strategy for BRAFV600E CRC patients. Citation Format: Mariah Geisen, Josiane weber-Tessmann, Courtney Kelson, Daheng He, Chi Wang, Abu Saleh Mosa Faisal, Jill Kolesar, Yekaterina Zaytseva. Inhibition of FASN postpones development of resistance to BRAF inhibitors in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4760.

Abstract 3008: Loss of Rab25 accompanied by NRAS activation cooperatively mediates the tumorigenic transformation in melanoma

Abstract Background: Ras oncogene activation is a common and often essential event in cancer development, including melanoma. Ras mutation is present in 20-30% of melanomas and remains an undrugged target. NRAS mutation is the most frequent Ras isoform activated in melanoma, with more than 80% cases of Q61 substitutions, and is closely associated with tumor aggressiveness. Rab25 is a ubiquitously expressed small GTPase protein with a context-dependent role in cancer pathophysiology. Its role remains undiscovered in the case of melanoma. Rab25 deep deletion can be seen in portions of melanoma in databases such as the Cancer Genome Atlas and Cbioportal. Interestingly, Rab25 has a consensus sequence homologous with the binding site of Q61 mutant Ras. We propose that the loss of Rab25 in melanocytes accompanied by Ras oncogenic activation are significant events in melanoma development, and restoration of Rab25 can potentially inhibit constitutively activated N-RAS; Rab25 serves as a tumor suppressor in melanoma genesis. Objectives: To discover the role of Rab25 in melanoma, we studied Rab25 expression via mRNA and protein levels in different melanoma cell lines and screened biopsy-proven melanoma samples through immunohistochemistry in 61 different melanoma sections. Further, to see the tumor suppressive mechanism of Rab25 in vitro, we transduced selected cell lines Rab25. Results: There is an 87% and 62.7% reduction in Rab25 mRNA level in NRASQ61R(SKMEL-2) and NRASQ61L type (WM1366) melanoma cell lines, respectively, also reflected in Rab25 protein level, and statistically significant at p-value <0.005, using student’s T-test. Further, Rab25, prominent in adjacent normal tissues, was significantly lost in biopsy-proven melanoma tissues, with an H-score ranging from 13.28 to 91.77% and 0.00 to-11.24 % for normal skin tissue and melanoma section, respectively, and a negative correlation of -0.350, indicating a promising association of Rab25 loss with the progression of melanoma. Conclusion: There is a loss of Rab25 in NRAS mutant melanoma with cancer progression. We saw a significant increase in Rab25 expression among the transduced sublines of NRAS mutant melanoma cell lines at the mRNA level, which will be further assessed through western blotting, cell proliferation assay, trans well migration assay, soft-agar colony formation assay to confirm NRAS mutant melanoma cell behavior. We will attempt to deliver a liposomally encapsulated six-peptide sequence to therapeutically inhibit the mutant NRAS. Citation Format: Samikshya Kandel, Krishna Rao. Loss of Rab25 accompanied by NRAS activation cooperatively mediates the tumorigenic transformation in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3008.

LncRNA NEAT1 contributes to multiple myeloma progression via upregulating ARPC5 by sponging miR-133a

IntroductionLong non-coding RNA (lncRNA) nuclear enriched abundant transcript 1 (NEAT1) is confirmed to be involved in regulation of the multiple myeloma (MM) process. However, its underlying molecular mechanism deserves further investigation.Material and methodsQuantitative real-time PCR was used to examine NEAT1, microRNA (miR)-133a and actin-related protein 2/3 complex subunit 5 (ARPC5) expression. Cell proliferation and apoptosis were evaluated by cell counting kit 8 assay, soft-agar colony formation assay and flow cytometry. Dual-luciferase reporter assay was used to confirm the interaction between miR-133a and NEAT1 or ARPC5. The localization of NEAT1 and miR-133a in MM cells was determined by fluorescent in situ hybridization assay. In addition, animal experiments were conducted to explore the effect of NEAT1 knockdown on MM tumor growth in vivo.ResultsNEAT1 had increased expression in MM patients and cells. NEAT1 knockdown could repress MM cell proliferation and enhance apoptosis. NEAT1 could sponge miR-133a, and miR-133a could target ARPC5. MiR-133a was lowly expressed and ARPC5 was highly expressed in MM patients. MiR-133a inhibitor could abolish the suppressive effect of NEAT1 knockdown on MM cell growth, and these effects also could be reversed by ARPC5 silencing.ConclusionsAnimal experiments showed that NEAT1 downregulation reduced MM tumor growth by regulating miR-133a/ARPC5 axis. NEAT1 facilitated MM progression through the regulation of miR-133a/ARPC5, which provided new evidence that NEAT1 was a potential therapeutic target for MM.

Protocol improvement and multisite validation of a digital soft agar colony formation assay for tumorigenic transformed cells intermingled in cell therapy products

Background aimsThe administration of human cell-processed therapeutic products (hCTPs) is associated with a risk of tumorigenesis due to the transformed cellular contaminants. To mitigate this risk, these impurities should be detected using sensitive and validated assays. The digital soft agar colony formation (D-SAC) assay is an ultrasensitive in vitro test for detecting tumorigenic transformed cells in hCTPs. Methods: In this study, we first evaluated the colony formation efficiency (CFE) precision of tumorigenic reference cells in positive control samples according to a previously reported D-SAC assay protocol (Protocol I) from multiple laboratories. However, the CFE varied widely among laboratories. Thus, we improved and optimized the test protocol as Protocol II to reduce variability in the CFE of tumorigenic reference cells. Subsequently, the improved protocol was validated at multiple sites. Human mesenchymal stromal cells (hMSCs) were used as model cells, and positive control samples were prepared by spiking them with HeLa cells. Results: Based on the previously reported protocol, the CFE was estimated using an ultra-low concentration (0.0001%) of positive control samples in multiple plates. Next, we improved the protocol to reduce the CFE variability. Based on the CFE results, we estimated the sample size as the number of wells (Protocol II) and assessed the detectability of 0.0001% HeLa cells in hMSCs to validate the protocol at multiple sites. Using Protocol I yielded low CFEs (mean: 30%) and high variability between laboratories (reproducibility coefficient of variance [CV]: 72%). In contrast, Protocol II, which incorporated a relatively high concentration (0.002%) of HeLa cells in the positive control samples, resulted in higher CFE values (mean: 63%) and lower variability (reproducibility CV: 18%). Moreover, the sample sizes for testing were estimated as the number of wells per laboratory (314–570 wells) based on the laboratory-specific CFE (42–76%). Under these conditions, all laboratories achieved a detection limit of 0.0001% HeLa cells in hMSCs in a predetermined number of wells. Moreover, colony formation was not observed in the wells seeded with hMSCs alone. ConclusionsThe D-SAC assay is a highly sensitive and robust test for detecting malignant cells as impurities in hCTPs. In addition, optimal assay conditions were established to test tumorigenic impurities in hCTPs with high sensitivity and an arbitrary false negative rate.

Antitumoral Potential of the Histone Demethylase Inhibitor GSK-J4 in Retinoblastoma.

The purpose of this study was to investigate the antitumor effects of GSK-J4 on retinoblastoma, as well as its related biological functions and molecular mechanisms. The antitumor effect of GSK-J4 on retinoblastoma was evaluated by in vitro and in vivo assays. CCK-8, EdU incorporation, and soft agar colony formation assays were performed to examine the effect of GSK-J4 on cell proliferation. Flow cytometry was used to evaluate the effect of GSK-J4 on the cell cycle and apoptosis. RNA-seq and Western blotting were conducted to explore the molecular mechanisms of GSK-J4. An orthotopic xenograft model was established to determine the effect of GSK-J4 on tumor growth. GSK-J4 significantly inhibited retinoblastoma cell proliferation both in vitro and in vivo, arrested the cell cycle at G2/M phase, and induced apoptosis. Mechanistically, GSK-J4 may suppress retinoblastoma cell growth by regulating the PI3K/AKT/NF-κB signaling pathway. The antitumor effects of GSK-J4 were noticeable in retinoblastoma and were at least partially mediated by PI3K/AKT/NF-κB pathway suppression. Our study provides a novel strategy for the treatment of retinoblastoma.

Paired-related homeobox 1 induces epithelial-mesenchymal transition in oesophageal squamous cancer.

It is unclear that paired-related homeobox 1 (PRRX1) induces epithelial-mesenchymal transition (EMT) in oesophageal cancer and the specific function of PRRX1 in oesophageal cancer metastasis. To assess the significance of PRRX1 expression and investigate the mechanism of EMT in oesophageal cancer metastasis. Detect the expression of PRRX1 by immunohistochemistry in oesophageal tumour tissues and adjacent normal oesophageal tissues; the PRRX1 short hairpin RNA (shRNA) or blank vector lentiviral gene delivery system was transfected into cells; cell proliferation assay, soft agar colony formation assays, cell invasion and migration assays and animal studies were used to observe cells biological characteristics In vitro and in vivo; XAV939 and LiCl were used to alter the activity of Wnt/β-catenin pathway. Immunofluorescence staining and western blot analysis were used to detect protein expression of EMT markers and Wnt/β-catenin pathway. PRRX1 is expressed at high levels in oesophageal cancer specimens and is closely related to tumour metastasis in patients with oesophageal cancer. Regulation of PRRX1 expression might exert obvious effects on cell proliferation, especially the migration and invasion of oesophageal cancer cells. Moreover, silencing PRRX1 expression using a shRNA produced the opposite effects. In addition, when PRRX1 was overexpressed, inhibition of the Wnt/β-catenin pathway with XAV939 negated the effect of PRRX1 on EMT, whereas when PRRX1 was downregulated, activation of the Wnt/β-catenin pathway with LiCl impaired the effect on EMT. PRRX1 is upregulated in oesophageal cancer is closely correlated with cancer metastasis. Additionally, PRRX1 induces EMT in oesophageal cancer metastasis through activation of Wnt/β-catenin signalling.

Abstract A096: RAS mutation status predicts activity of the SHP2 inhibitor TNO155 in RAS-pathway driven fusion-negative rhabdomyosarcoma

Abstract Rhabdomyosarcomas (RMS) are the most common pediatric soft tissue sarcoma. Fusion-negative RMS (FN-RMS, the subset lacking FOXO1 translocations), are characterized by oncogenic point mutations in genes that regulate activity of the RAS/ERK signaling pathway. Clinically available agents that directly target oncogenic RAS in cancer have only recently become FDA approved, and their activity is limited to cancers with G12C mutations in KRAS. Alternate molecular strategies to target RAS activation in cancer include inhibitors of RAS regulatory proteins, including the nonreceptor tyrosine phosphatase SHP2. SHP2 regulates RAS signaling by dephosphorylating receptor tyrosine kinases (RTKs), preventing effective RAS GTPase-activating protein (GAP) recruitment. Additionally, SHP2 recruits SOS1, a RAS guanine nucleotide exchange factor (GEF), promoting RAS activation. Novel small molecule inhibitors of SHP2 are now in clinical trials in several cancer types. We set out to determine whether SHP2 inhibitors demonstrate preclinical activity in models of RAS-pathway driven FN-RMS. We hypothesize that specific oncogenic mutations in HRAS, NRAS and KRAS, affecting codons 12, 13 and 61, will determine SHP2 inhibitor sensitivity in models of FN-RMS, and that sensitivity will be determined based on the interactions between RAS and RAS-GAPs/RAS-GEFs. We tested the effects of SHP2 inhibition on ERK pathway signaling, using the small molecule inhibitor TNO155, in a panel of FN-RMS cell lines with varied RAS mutation status. In FN-RMS cells with Q61-mutant HRAS or NRAS, phospho-MEK and phospho-ERK were unchanged, while in cells with RTK-amplification and wild-type RAS, or those with G12 or G13-mutant HRAS we observed reductions in p-MEK and p-ERK following treatment with TNO155. Reduced 2-D and 3-D cell growth were also observed in WT and G12/G13-mutant HRAS cell lines, but not those with Q61 mutations, using both high throughput proliferation assays and soft-agar colony formation assays, respectively. Responses in sensitive cells were modest and cytostatic at best, suggesting single agent SHP2i may have insufficient activity in these tumors. RNAseq analysis of TNO155-sensitive RAS-WT RMS cells reveals a set of differentially expressed genes that may inform the selection of agents for combination therapy. In addition, a subset of FN-RMS cells also harbor activating mutations in FGFR and PI3K-associated genes, and we therefore tested whether TNO155 might synergize with FGFR or PI3K small-molecule inhibitors and found additive effects between TNO155, the PI3Ki alpelisib, and the pan-FGFRi erdafitinib. In conclusion, FN-RMS cells driven by RTK-amplification, or by RAS G12 or G13 mutations, are sensitive to TNO155 while cells driven by RAS Q61 mutations are not. Additionally, FN-RMS cells treated with TNO155 may rely on the PI3K pathway for adaptive resistance, and combination treatment involving TNO155 and FGFR or PI3K inhibitors may reduce tumor growth with better efficacy than either compound alone. Citation Format: Andrew Baker, Patience Odeniyide, Alyza Skaist, Alla Lisok, Elana Fertig, Jiawan Wang, Christine Pratilas. RAS mutation status predicts activity of the SHP2 inhibitor TNO155 in RAS-pathway driven fusion-negative rhabdomyosarcoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A096.

ITGB3 Promotes the Progression of JAK2V617F-Positive Myeloproliferative Neoplams By Regulating Calreticulin to Inhibit Cellular Autophagy

O bjectives: Integrins are integral cell-surface proteins composed of an alpha chain and a beta chain, of which integrin beta 3 (ITGB3) was known to participate in cell adhesion as well as cell-surface mediated signaling and plays vital roles in different tumors. However, the role of ITGB3 in JAK2V617F-positive classical myeloproliferative neoplasms (cMPNs) remians unclear, and the aim of this study was to explore the effect of ITGB3 on cellular autophagy in JAK2V617F-positive cMPN cell line and its underlying mechanisms. Methods: ITGB3 expression in bone marrows from JAK2V617F-positive cMPN patients compared to normal controls were detected by quantitative reverse-transcription PCR (qPCR) and further validated by western blotting. Cell models of cMPNs were constructed using human HEL cell lines carrying JAK2V617F mutation and cytokine-dependent wild type BaF3 cells (Jak2 V617F BaF3 cells) with ectopic expression of Jak2 V617F seperately to perform phenotype and mechanism investigation. The putative targets of STAT5a on the promoters of ITGB3 were disclosed by public databases and a dual-luciferase reporter assay and chromatin immunoprecipitation assay (ChIP) was used to analyze the binding of STAT5a to ITGB3 promoter region. To observe the role of ITGB3 played in cMPN progression, stable-transfected HEL cell line with ITGB3 knockdown or overexpression were established by lentivirus packaging and cell transfection. Cell phenotype including cell proliferation, apoptosis and cell cycle distribution were detected by soft agar colony formation assay, BrdU staining, flow cytometry and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays. The variations of autophagic flux after ITGB3 dysregulation in HEL cells was analyzed using mRFP-GFP-LC3 adenovirus transfection and fluorescence microscopy, and LC3-II/I ratio as well as autophagy-related protein were detected by western blotting. Transmission electron microscopy was used to observe the formation of intracellular autophagosomes and autophagic lysosomes. RNA sequencing and GSEA analysis were applied to predict the downstream pathway and putative targets of ITGB3. Western blotting, immunofluorescence and qPCR were performed to detect the transcription and expression levels of genes. Results: 1.Western blotting and qRT-PCR detection showed increased expression of ITGB3 in bone marrow of JAK2V617F-positive cMPN patients compared to normal controls (A-B). Also, the evaluation of ITGB3 in a variety of myeloid tumor cell lines (HEL, HL60, K562, KG-1a) and wild-type BaF3 cells as well as Jak2 V617F BaF3 cells revealed that ITGB3 protein level is the highest in HEL (which harboring the JAK2V617F) and Jak2 V617F BaF3 cells (C-F). 2. Dual-luciferase reporter assay showed that STAT5a significantly induced the transcription activities of promoters of ITGB3 and inhibition of STAT5a reduced the transcription activities of promoters of ITGB3 (G-H). ITGB3 knockdown (I) in HEL cells reduced the proliferation capacity (J-L), promoted early apoptosis (M-O) and caused cell cycle arrest (P-Q). While ITGB3 overexpression in HEL cells (R) reduced the cell cycle arrest (S) and apoptosis (T-U). 3. Autophagic flux analysis showed that ITGB3 knockdown promoted the autophagic flux in HEL cells (V-X). Inhibition of ITGB3 increases the synthesis and degradation of autophagosomes and accelerates the process of cellular autophagy (Y-Z). Further identification of DEGs in ITGB3-dysregulated HEL cells (AA-AB) reveals that ITGB3 inhibits the expression of calreticulin (AC-AE), and ITGB3 might be involved in the regulation of autophagy in HEL cells through the regulation of calreticulin. Conclusions: 1. ITGB3 is significantly activated in HEL cell lines carrying the JAK2V617F mutation. 2. ITGB3 protein acts as a pro-oncogene in JAK2V617F-positive myeloproliferative neoplams progression by regulating calreticulin to inhibit autophagy, which deserves more in-depth study in JAK2V617F mutation-positive cMPNs.

Inhibition of GMA-induced ubiquitination process in 16HBE malignantly transformed cells on protein CD44 and MMP14

To observe the effect of the ubiquitination process on the expression of CD44 antigen(CD44) and matrix metalloproteinase-14(MMP14) in human bronchial epithelial(16HBE) malignantly transformed cells induced by glycidyl methacrylate(GMA). Successfully resuscitated 16HBE cells were cultured using a final concentration of 8 μg/mL GMA as the treatment group and 1 μg/mL dimethyl sulfoxide as the solvent control group, each time stained for 72 h, and then stained again after an interval of 24 h. After repeating the staining three times, the cells were cultured in passages respectively. The 40th generation(P40) GMA-treated group and the same-generation solvent control group were subjected to soft agar colony formation assay and concanavalin A(ConA) agglutination test to confirm that the 40th generation of GMA-induced malignant transformed 16HBE cells possessed malignant transformed cell characteristics.5, 10, 20, 40, 60 μmol/L anacardic acid were used to inhibit the ubiquitination process of GMA-induced malignant transformed 16HBE cells. The protein expression of CD44 and MMP14 were detected by western blotting, while the transcript levels of CD44, MMP14, and TFAP2A were assessed by real-time fluorescence quantitative PCR(qPCR). (1) In the soft agar colony formation assay, the number of clones formed by the cells in the solvent control group was 22, and the number of clones created by the malignantly transformed cells in the GMA-treated group was 208. In the ConA agglutination test, the cells in the solvent control group were uniformly dispersed in ConA solution, and no obvious agglutination occurred for 30 min, whereas the cells in the GMA-treated group were agglutinated in the 5th min, and the agglutinated cells were larger and more rapidly agglutinated. The agglomerates were more significant and faster, and the sensitivity of agglutination was increased. (2) After differential inhibition of GMA-induced ubiquitination in malignantly transformed 16HBE cells, the expression levels of CD44 and MMP14 were reduced in GMA-induced malignantly transformed 16HBE cells compared with the control group(P<0.05). The transcript levels of MMP14 and CD44 decreased with increasing inhibitor concentration(P<0.05), and the transcript levels of the upstream transcription factor TFAP2A were also simultaneously reduced(P<0.05). Inhibition of the cellular ubiquitination process mediates the down-regulation of protein expression and transcriptional expression of CD44 and MMP14 in GMA-induced malignantly transformed 16HBE cells.

Effects of MCU-mediated Ca2+ Homeostasis on Ovarian Cancer Cell SKOV3 Proliferation, Migration and Transformation.

Atlas human proteomics database showed MCU as highly expressed in various tumor tissues, especially in ovarian cancer. Rare studies on the role of MCU and its regulation in ovarian cancer have been reported. The objective of this study was to determine role of MCU in ovarian cancer cell SKOV3 proliferation, migration, and transformation, and explore the possible mechanism. MCU siRNA on lentiviral particles were stably transfected into SKOV3 cells. CCK-8 assay was performed to analyze cell proliferation. Soft-agar colony formation assay was employed to evaluate tumorigenesis. Western blot and immunohistochemistry analyses were performed to evaluate the expression of MCU, MICU1 and phosphorylate of Akt in the ovarian cancer cell and tissue specimens. Scratch assay was combined with trans-well plates assay to detect the migration ability of cancer cells. The ROS production and Ca2+ expression were also determined. MCU expression was significantly higher in ovarian cancer tissues than normal tissues. MCU silencing decreased SKOV3 cell proliferation, migration, and transformation. ROS production was decreased after MCU silencing, depending on disturbed Ca2+ homeostasis. MICU1 expression has been found to be decreased and phosphorylation of Akt increased when MCU was silenced. Down-regulation of MCU inhibited SKOV3 cell proliferation, migration, and transformation via disturbing Ca2+ homeostasis and decreasing ROS production. MICU1 and phosphorylation of Akt are associated with MCU-mediated ovarian cancer malignancy.

LncRNA RP11-93B14.5 promotes gastric cancer cell growth through PI3K/AKT signaling pathway.

Emerging evidence indicates that long non-coding RNA (lncRNA) RP11-93B14.5 facilitates tumor progression in variety of malignancies. The present study proposed to study the functional effect of lncRNA RP11-93B14.5 in gastric cancer (GC) as well as the underlying mechanism. Bioinformatics analysis was utilized to analyze lncRNA expression in GC tissues. siRNA was used for knockdown of RP11-93B14.5 in GC cells MKN45 and KATO III. The stable knockdown cell lines were constructed by CRISPR-Cas9. Cell counting kit-8 (CCK-8) assay and soft agar colony formation assay were used to analyze GC cell viability. Flow cytometry analysis was performed to analyze the cell cycle distribution of MKN45 and KATO III. RNA sequencing (RNA-seq) was employed to detect differential genes after transfection with siRP11-93B14.5. Quantitative PCR (Q-PCR) was used to examine gene expression in GC cell lines. Western-blot assay was used to measure protein levels. RNA fluorescent in situ hybridization (FISH) was conducted for lncRNA cellular location and expression. Based on the Cancer Genome Atlas (TCGA)and Genotype-Tissue Expression (GTEx) database, RP11-93B14.5 was upregulated in GC tissue, which was also verified in GC cell lines in comparison to the normal gastric epithelial HFE145 cells. Knockdown of RP11-93B14.5 decreased cell viability and the colony number of MKN45 and KATO III cells, and altered cell cycle distribution in vitro. RNA-seq analysis revealed RP11-93B14.5 may modulate genes expression of S100A2 and TIMP2in MKN45 and KATO III cells. Mechanistically, RP11-93B14.5 may drive the progression of GC via S100A2 related-PI3K/AKT signaling pathway. LncRNA RP11-93B14.5 knockdown alleviated the malignant phenotypes of GC cells through regulating PI3K/AKT. Our results provide evidence for the role of lncRNAs in regulating tumor progression.

Non-chemotherapy adjuvant agents in TP53 mutant Ewing sarcoma

Ewing sarcoma (EWS) is a malignant tumor arising in bone or soft tissue that occurs in adolescent and young adult patients as well as adults later in life. Although non-metastatic EWS is typically responsive to treatment when newly diagnosed, relapsed cases have an unmet need for which no standard treatment approach exists. Recent phase III clinical trials for EWS comparing 7 vs 5 chemotherapy drugs have failed to improve survival. To extend the durability of remission for EWS, we investigated 3 non-chemotherapy adjuvant therapy drug candidates to be combined with chemotherapy. The efficacy of these adjuvant drugs was investigated via anchorage-dependent growth assays, anchorage-independent soft-agar colony formation assays and EWS xenograft mouse models. Enoxacin and entinostat were the most effective adjuvant drug in both long-term in vitro and in vivo adjuvant studies. In the context that enoxacin is an FDA-approved antibiotic, and that entinostat is an investigational agent not yet FDA-approved, we propose enoxacin as an adjuvant drug for further preclinical and clinical investigation in EWS patients.

Synergistic Chemopreventive Effects of a Novel Combined Plant Extract Comprising Gallic Acid and Hesperidin on Colorectal Cancer.

Colorectal cancer (CRC) is the third most common cancer with a high mortality rate worldwide. Although gallic acid and hesperidin exert anticancer activity, synergistic effects of gallic acid and hesperidin against CRC remain elusive. This study aims to investigate the therapeutic mechanism of a novel combination of gallic acid and hesperidin against CRC cell growth, including cell viability, cell-cycle-associated proteins, spheroid formation, and stemness. Gallic acid and hesperidin derived from Hakka pomelo tea (HPT) were detected by colorimetric methods and high-performance liquid chromatography using ethyl acetate as an extraction medium. CRC cell lines (HT-29 and HCT-116) treated with the combined extract were investigated in our study for cell viability (trypan blue or soft agar colony formation assay), cell cycle (propidium iodide staining), cell-cycle-associated proteins (immunoblotting), and stem cell markers (immunohistochemistry staining). Compared with other extraction methods, HPT extraction using an ethyl acetate medium exerts the most potent effect on inhibiting HT-29 cell growth in a dose-dependent manner. Furthermore, the treatment with combined extract had a higher inhibitory effect on CRC cell viability than gallic acid or hesperidin alone. The underlying mechanism was involved in G1-phase arrest and Cip1/p21 upregulation that could attenuate HCT-116 cell proliferation (Ki-67), stemness (CD-133), and spheroid growth in a 3D formation assay mimicking in vivo tumorigenesis. Gallic acid and hesperidin exert synergistic effects on cell growth, spheroids, and stemness of CRC and may serve as a potential chemopreventive agent. Further testing for the safety and effectiveness of the combined extract in large-scale randomized trials is required.

Similar deamination activities but different phenotypic outcomes induced by APOBEC3 enzymes in breast epithelial cells.

APOBEC3 (A3) enzymes deaminate cytosine to uracil in viral single-stranded DNA as a mutagenic barrier for some viruses. A3-induced deaminations can also occur in human genomes resulting in an endogenous source of somatic mutations in multiple cancers. However, the roles of each A3 are unclear since few studies have assessed these enzymes in parallel. Thus, we developed stable cell lines expressing A3A, A3B, or A3H Hap I using non-tumorigenic MCF10A and tumorigenic MCF7 breast epithelial cells to assess their mutagenic potential and cancer phenotypes in breast cells. The activity of these enzymes was characterized by γH2AX foci formation and in vitro deamination. Cell migration and soft agar colony formation assays assessed cellular transformation potential. We found that all three A3 enzymes had similar γH2AX foci formation, despite different deamination activities in vitro. Notably, in nuclear lysates, the in vitro deaminase activity of A3A, A3B, and A3H did not require digestion of cellular RNA, in contrast to that of A3B and A3H in whole-cell lysates. Their similar activities in cells, nonetheless, resulted in distinct phenotypes where A3A decreased colony formation in soft agar, A3B decreased colony formation in soft agar after hydroxyurea treatment, and A3H Hap I promoted cell migration. Overall, we show that in vitro deamination data do not always reflect cell DNA damage, all three A3s induce DNA damage, and the impact of each is different.

CPEB2 enhances cell growth and angiogenesis by upregulating ARPC5 mRNA stability in multiple myeloma

BackgroundThe process of multiple myeloma (MM) is the result of the combined action of multiple genes. This study aims to explore the role and mechanism of cytoplasmic polyadenylation element binding protein2 (CPEB2) in MM progression.MethodsThe mRNA and protein expression levels of CPEB2 and actin-related protein 2/3 complex subunit 5 (ARPC5) were assessed by quantitative real-time PCR and western blot analysis. Cell function was determined by cell counting kit 8 assay, soft-agar colony formation assay, flow cytometry and tube formation assay. Fluorescent in situ hybridization assay was used to analyze the co-localization of CPEB2 and ARPC5 in MM cells. Actinomycin D treatment and cycloheximide chase assay were performed to assess the stability of ARPC5. The interaction between CPEB2 and ARPC5 was confirmed by RNA immunoprecipitation assay.ResultsCPEB2 and ARPC5 mRNA and protein expression levels were upregulated in CD138+ plasma cells from MM patients and cells. CPEB2 downregulation reduced MM cell proliferation, angiogenesis, and increased apoptosis, while its overexpression had an opposite effect. CPEB2 and ARPC5 were co-localized at cell cytoplasm and could positively regulate ARPC5 expression by mediating its mRNA stability. ARPC5 overexpression reversed the suppressive effect of CPEB2 knockdown on MM progression, and it knockdown also abolished CPEB2-promoted MM progression. Besides, CPEB2 silencing also reduced MM tumor growth by decreasing ARPC5 expression.ConclusionOur results indicated that CPEB2 increased ARPC5 expression through promoting its mRNA stability, thereby accelerating MM malignant process.

Deubiquitinating enzyme MINDY1 is an independent risk factor for the maintenance of stemness and poor prognosis in liver cancer cells

Objective: To explore the key deubiquitinating enzymes that maintain the stemness of liver cancer stem cells and provide new ideas for targeted liver cancer therapy. Methods: The high-throughput CRISPR screening technology was used to screen the deubiquitinating enzymes that maintain the stemness of liver cancer stem cells. RT-qPCR and Western blot were used to analyze gene expression levels. Stemness of liver cancer cells was detected by spheroid-formation and soft agar colony formation assays. Tumor growth in nude mice was detected by subcutaneous tumor-bearing experiments. Bioinformatics and clinical samples were examined for the clinical significance of target genes. Results: MINDY1 was highly expressed in liver cancer stem cells. The expression of stem markers, the self-renewal ability of cells, and the growth of transplanted tumors were significantly reduced and inhibited after knocking out MINDY1, and its mechanism of action may be related to the regulation of the Wnt signaling pathway. The expression level of MINDY1 was higher in liver cancer tissues than that in adjacent tumors, which was closely related to tumor progression, and its high expression was an independent risk factor for a poor prognosis of liver cancer. Conclusion: The deubiquitinating enzyme MINDY1 promotes stemness in liver cancer cells and is one of the independent predictors of poor prognosis in liver cancer.

Rhein Induces Oral Cancer Cell Apoptosis and ROS via Suppresse AKT/mTOR Signaling Pathway In Vitro and In Vivo.

Oral cancer remains the leading cause of death worldwide. Rhein is a natural compound extracted from the traditional Chinese herbal medicine rhubarb, which has demonstrated therapeutic effects in various cancers. However, the specific effects of rhein on oral cancer are still unclear. This study aimed to investigate the potential anticancer activity and underlying mechanisms of rhein in oral cancer cells. The antigrowth effect of rhein in oral cancer cells was estimated by cell proliferation, soft agar colony formation, migration, and invasion assay. The cell cycle and apoptosis were detected by flow cytometry. The underlying mechanism of rhein in oral cancer cells was explored by immunoblotting. The in vivo anticancer effect was evaluated by oral cancer xenografts. Rhein significantly inhibited oral cancer cell growth by inducing apoptosis and S-phase cell cycle arrest. Rhein inhibited oral cancer cell migration and invasion through the regulation of epithelial-mesenchymal transition-related proteins. Rhein induced reactive oxygen species (ROS) accumulation in oral cancer cells to inhibit the AKT/mTOR signaling pathway. Rhein exerted anticancer activity in vitro and in vivo by inducing oral cancer cell apoptosis and ROS via the AKT/mTOR signaling pathway in oral cancer. Rhein is a potential therapeutic drug for oral cancer treatment.