Decreased Colony Formation Research Articles

Retraction Note to: Loss of PDEF, a prostate-derived Ets factor is associated with aggressive phenotype of prostate cancer: Regulation of MMP 9 by PDEF

Prostate-derived Ets factor (PDEF) is expressed in tissues of high epithelial content including prostate, although its precise function has not been fully established. Conventional therapies produce a high rate of cure for patients with localized prostate cancer, but there is, at present, no effective treatment for intervention in metastatic prostate cancer. These facts underline the need to develop new approaches for early diagnosis of aggressive prostate cancer patients, and mechanism based anti-metastasis therapies that will improve the outlook for hormone-refractory prostate cancer. In this study we evaluated role of prostate-derived Ets factor (PDEF) in prostate cancer. We observed decreased PDEF expression in prostate cancer cell lines correlated with increased aggressive phenotype, and complete loss of PDEF protein in metastatic prostate cancer cell lines. Loss of PDEF expression was confirmed in high Gleason Grade prostate cancer samples by immuno-histochemical methods. Reintroduction of PDEF profoundly affected cell behavior leading to less invasive phenotypes in three dimensional cultures. In addition, PDEF expressing cells had altered cell morphology, decreased FAK phosphorylation and decreased colony formation, cell migration, and cellular invasiveness. In contrast PDEF knockdown resulted in increased migration and invasion as well as clonogenic activity. Our results also demonstrated that PDEF downregulated MMP9 promoter activity, suppressed MMP9 mRNA expression, and resulted in loss of MMP9 activity in prostate cancer cells. These results suggested that loss of PDEF might be associated with increased MMP9 expression and activity in aggressive prostate cancer. To confirm results we investigated MMP9 expression in clinical samples of prostate cancer. Results of these studies show increased MMP9 expression correlated with advanced Gleason grade. Taken together our results demonstrate decreased PDEF expression and increased MMP9 expression during the transition to aggressive prostate cancer. These studies demonstrate for the first time negative regulation of MMP9 expression by PDEF, and that PDEF expression was lost in aggressive prostate cancer and was inversely associated with MMP9 expression in clinical samples of prostate cancer. Based on these exciting results, we propose that loss of PDEF along with increased MMP9 expression should serve as novel markers for early detection of aggressive prostate cancer.

Changes in Wnt and TGF-β Signaling Mediate the Development of Regorafenib Resistance in Hepatocellular Carcinoma Cell Line HuH7.

Hepatocellular carcinoma (HCC) is an aggressive, chemo resistant neoplasm with poor prognosis and limited treatment options. Exploring activated pathways upon drug treatment can be used to discover more effective anticancer agents to overcome therapy resistance and enhance therapeutic outcomes for patients with advanced HCC. Human tumor-derived cell lines recapitulate HCC diversity and are widely used for studying mechanisms that drive drug resistance in HCC. In this study, we show that regorafenib treatment activates Wnt/β-catenin signaling only in hepatoblast-like HCC cell lines and induces enrichment of markers associated with hepatic stem/progenitor cells. Moreover, activation of Wnt/β-catenin signaling via Wnt3a/R-Spo1 treatment protects these cells from regorafenib induced apoptosis. On the other hand, regorafenib resistant cells established by long-term regorafenib treatment demonstrate diminished Wnt/β-catenin signaling activity while TGF-β signaling activity of these cells is significantly enhanced. Regorafenib resistant cells (RRCs) also show increased expression of several mesenchymal genes along with an induction of CD24 and CD133 cancer stem cell markers. Moreover, regorafenib resistant cells also exhibit significantly augmented in vitro and in vivo migration capacity which could be reversed by TGF-β type 1 receptor (TGFb -R1) inhibition. When combined with regorafenib treatment, TGFβ-R1 inhibition also significantly decreased colony formation ability and augmented cell death in resistant spheroids. Importantly, when we knocked down TGFβ-R1 using a lentiviral plasmid, regorafenib resistant cells entered senescence indicating that this pathway is important for their survival. Treatment of RRCs with TGFβ-R1 inhibitor and regorafenib significantly abolished pSTAT3, pSMAD2 and pERK (44/42) expression suggesting the involvement of both canonical and non-canonical pathways. In conclusion, our data suggest that HCC tumors with aberrant activation in the Wnt/β-catenin pathway, might have higher intrinsic regorafenib resistance and the inhibition of this pathway along with regorafenib administration might increase regorafenib-induced cell death in combinational therapies. However, to resolve acquired regorafenib resistance developed in HCC patients, the combined use of TGF-β pathway inhibitors and Regorafenib constitute a promising approach that can increase regorafenib sensitization and prevent tumor recurrence.

Local anesthetic bupivacaine inhibits proliferation and metastasis of hepatocellular carcinoma cells via suppressing PI3K/Akt and MAPK signaling.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide. Retrospective studies suggest that using local/regional anesthetic (LA/RA) is associated with better outcomes in primary HCC patients. In this study, we evaluated the effects of LA/RA bupivacaine in HCC cells and the underlying molecular mechanisms. The biological functions of bupivacaine in HCC cells were evaluated by transcriptome RNA sequencing, cell viability assay, bromodeoxyuridine incorporation assay, colony formation assay, flow cytometry, western blot, wound healing assay, transwell cell migration assay, tumor xenograft formation, and lung metastasis assay. Bupivacaine suppressed proliferation and induced apoptosis of HepG2 and SNU-449 cells in a time- and dose-dependent manner. Bupivacaine treatment also decreased colony formation, migration, and invasion of HepG2 and SNU-449 cells. In mouse models, bupivacaine repressed tumor xenograft growth and lung metastasis of HepG2 cells. Transcriptome sequencing of HepG2 cells suggested that PI3K/Akt and MAPK signaling pathways were suppressed by bupivacaine treatment. In western blot analysis, bupivacaine reduced the expression of total and phosphorylated Akt, mTOR, and MAPK. Furthermore, reactivated PI3K/Akt and MAPK signaling by EGF or NRG1 partially reversed the effects of bupivacaine on cell growth, colony formation, and invasion of HCC cells. Local anesthetic bupivacaine suppressed proliferation, migration and invasion, and induced apoptosis of HCC cells. Our results provided novel insights into the local anesthetic bupivacaine in the therapy of HCC patients.

Leucyl-tRNA synthetase 1 is required for proliferation of TSC-null cells

Uncontrolled cell proliferation associated with cancer depends on the functional abrogation of at least one of tumor suppressor. In response to nutrient cue, tuberous sclerosis complex (TSC) works as a tumor suppressor which inhibits cell growth via negative regulation of the mammalian target of rapamycin complex (mTORC1). However, the regulation mechanism of nutrient-dependent cell proliferation in TSC-null cells remains unclear. Here, we demonstrate that leucine is required for cell proliferation through the activation of leucyl-tRNA synthetase (LARS1)-mTORC1 pathway in TSC-null cells. Cell proliferation and survival were attenuated by LARS1 knock-down or inhibitors in TSC-null cells. In addition, either rapamycin or LARS1 inhibitors significantly decreased colony formation ability while their combined treatment drastically attenuated it. Taken together, we suggest that LARS1 inhibitors might considered as novel tools for the regression of tumor growth and proliferation in TSC-null tumor cells which regrow upon discontinuation of the mTORC1 inhibition.

PSMC2/CCND1 axis promotes development of ovarian cancer through regulating cell growth, apoptosis and migration

Ovarian cancer is known as one of the most common malignancies of the gynecological system, whose treatment is still not satisfactory because of the unclear understanding of molecular mechanism. PSMC2 is an essential component of 19 S regulatory granules in 26 S proteasome and its relationship with ovarian cancer is still not clear. In this study, we found that PSMC2 was upregulated in ovarian cancer tissues, associated with tumor grade and could probably predict poor prognosis. Knocking down the endogenous PSMC2 expression in ovarian cancer cells could decrease colony formation ability, cell motility and cell proliferation rate, along with increasing cell apoptosis rate. Cells models or xenografts formed by cells with relatively lower expression of PSMC2 exhibited weaker oncogenicity and slower growth rate in vivo. Moreover, gene microarray was used to analyze the alteration of gene expression profiling of ovarian cancer induced by PSMC2 knockdown and identify CCND1 as a potential downstream of PSMC2. Further study revealed the mutual regulation between PSMC2 and CCND1, and demonstrated that knockdown of CCND1 could enhance the regulatory effects induced by PSMC2 knockdown and overexpression of CCND1 reverses it. In summary, PSMC2 may promote the development of ovarian cancer through CCND1, which may predict poor prognosis of ovarian cancer patients.

Abstract 1408: PlexinD1 drives enzalutamide-resistant neuroendocrine prostate cancer development and progression

Abstract Background: Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer (PC) arising as a consequence of more potent targeting of the androgen receptor (AR) pathway in castration-resistant prostate cancer (CRPC). Recently, the transition from CRPC to NEPC occurs more frequently due to the widespread use of highly potent antiandrogens such as enzalutamide (ENZ). This study sought to investigate the functional role of PlexinD1 in ENZ-induced NEPC and dissect the underlying molecular mechanism. Methods: The PlexinD1 expression levels and its association with neuroendocrine differentiation were determined in publicly available multi-omics datasets and clinical specimens. Human PC stable cell lines with augmentation or silencing of PlexinD1 expression were established and assayed for cell proliferation, colony formation and neuroendocrine marker gene expression by Western blotting and RT-qPCR analyses. Results: PlexinD1 expression was elevated in hormone-refractory PC compared to hormone-sensitive PC and further in NEPC compared to CRPC, which was paralleled by a positive correlation between PlexinD1 expression and NEPC score and an inverse relationship of PlexinD1 levels with AR score, by a survey of multiple independent clinical datasets. The association of PlexinD1 with different subtypes and disease status of PC will also be explored in our own patient cohorts by quantitative immunohistochemical analysis. The PlexinD1 mRNA levels were induced in androgen-dependent LNCaP cells grown in hormone-depleted media for 3 days and in C4-2B cells treated with 10 µM ENZ for 5 days. PlexinD1 was also increased in ENZ-resistant C4-2B (C4-2BENZR) cells at both mRNA and protein levels. shRNA-mediated knockdown of PlexinD1 in neuroendocrine-like PC cells, including C4-2BENZR, 22Rv1, PC-3 and DU145, suppressed cell proliferation by up to more than 50%, which was corroborated by decreased colony formation. PlexinD1 silencing in C4-2BENZR and 22Rv1 cells led to reduced expression of the NE marker chromogranin A (CHGA). The effects of PlexinD1 on NEPC tumor growth and neuroendocrine marker expression will be further examined in xenograft mouse models. Collectively, our data indicate that PlexinD1 plays an essential role supporting the acquisition of a neuroendocrine phenotype in PC cells and driving NEPC cell survival and growth. Conclusion: Our study suggests that PlexinD1 promotes the progression of CRPC to NEPC under ENZ pressure and subsequent emergence of resistance to ENZR possibly through induction of neuroendocrine traits, which provides new insights into further developing PlexinD1 as a new biomarker and therapeutic target in NEPC. Funding provided by Department of Defense PCRP grant W81XWH-19-1-0279, NIH/NCI grant R37CA233658, and the WSU start-up fund (to B.W.) Citation Format: Jing Wang, Jingjing Li, Boyang Wu. PlexinD1 drives enzalutamide-resistant neuroendocrine prostate cancer development and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1408.

Abstract 1213: PDE10A as a novel target to suppress Wnt/β-catenin signaling and other oncogenic pathways in ovarian cancer

Abstract Objective: A leading theory for ovarian carcinogenesis proposes that the inflammation associated with incessant ovulation is a driver of oncogenesis. Consistent with this theory, epidemiological studies have shown that nonsteroidal anti-inflammatory drugs (NSAIDs) decrease ovarian cancer risk. Previous studies suggest, however, that the antineoplastic activity of NSAIDs does not require the traditional cyclooxygenase (COX) enzyme inhibition, and rather may be exerted through phosphodiesterase (PDE) inhibition. PDEs represent a potentially unique chemopreventive target for ovarian cancer given that ovulation is regulated by cyclic nucleotide signaling. This study evaluates the effects of phosphodiesterase 10A (PDE10A) inhibition as a novel chemopreventive and therapeutic approach for ovarian cancer. Methods: We investigated the effects of PDE10A small molecule inhibitors, Pf-2545920 and MCI-030 (a novel non-COX inhibitory NSAID-derivative), and PDE10A knockout by CRISPR/Cas9 gene editing in various ovarian cancer cell lines using in vitro assays that measured cell proliferation, cell viability, cell cycle arrest, apoptosis, cell migration and invasion. Downstream signaling pathways affected by PDE10A inhibition and gene knockout (KO) were assessed by western-blotting, confocal microscopy, and RNA sequencing. Results: Analysis of The Cancer Genome Atlas (TCGA) ovarian cancer database and an institutional cohort of ovarian cancer patients revealed that high PDE10A mRNA expression was associated with significantly worse overall survival. PDE10A expression was also positively correlated with oncogenic and inflammatory signaling pathways in the TCGA dataset. PDE10A inhibition with Pf-2545920 or MCI-030 decreased ovarian cancer cell proliferation, while inducing cell cycle arrest and apoptosis. Using inhibitors to block PKA (H89) and PKG (KT5823) kinase activity, we demonstrated that the pro-apoptotic effects of PDE10A inhibition were mediated by activation of cGMP/PKG and cAMP/PKA signaling. SKOV3 and OV-90 PDE10A KO cells showed decreased colony formation, cell proliferation, and migration and invasion properties compared to their wild-type (WT) counterparts. Moreover, PDE10A inhibition decreased Wnt-induced β-catenin nuclear translocation as well as decreased EGF-mediated activation of RAS/MAPK and AKT pathways in ovarian cancer cells. RNA sequencing of SKOV3 PDE10A KO clones revealed that pathways associated with cancer, Wnt and TGF-β signaling were downregulated compared to WT control cells. Conclusions: Our data demonstrate that PDE10A has pro-tumorigenic effects in ovarian cancer cells and its high expression in ovarian cancer patients is associated with poor prognosis. Altogether, our results warrant future studies of PDE10A as a novel target for ovarian cancer chemoprevention and/or treatment. Citation Format: Rebecca M. Barber, Elaine Gavin, Alla Musiyenko, Wito Richter, Kevin J. Lee, Annelise Wilhite, Joel F. Andrews, Steve McClellan, Ileana Aragon, Antonio Ward, Xi Chen, Adam Keeton, Kristy Berry, Gary A. Piazza, Jennifer M. Scalici, Luciana Madeira da Silva. PDE10A as a novel target to suppress Wnt/β-catenin signaling and other oncogenic pathways in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1213.

Combination Treatment with an MDM2 Antagonist and a BET Inhibitor Targets Both Myelofibrosis Hematopoietic Stem/ Progenitor Cells and Their Tumor Promoting Microenvironment

Introduction: Myelofibrosis (MF) arises at the level of hematopoietic stem and progenitor cells (HSC/HPC) due to a sequence of mutational events that results in increased JAK/STAT signaling. JAK2 has served as a target for drug development and has led to the development of several novel small molecule inhibitors. Clinical trials of these agents have revealed their ability to decrease the degree of splenomegaly and to improve systemic symptoms of PMF patients, but not to substantially affect the progeny of the malignant clone or the rate of disease progression. MF HSC/HPCs and their progeny are known to elaborate a number pro-inflammatory mediators including lipocalin (LCN2) (Lu et al, Blood, 2015), and IL-8 which are capable of creating a tumor promoting microenvironment. LCN2 promotes osteoblast formation and marrow fibrosis (Lu et al, Blood, 2013) while IL8 promotes endothelial cell proliferation. We have previously reported that plasma LCN2 levels are increased in MF patients (Lu et al, ASH, 2016) and show now that plasma IL-8 levels are also dramatically increased (Figure 1). Furthermore the levels of IL-8 as shown by IHC were increased in splenic myeloid cells especially megakaryocytes and the IL-8 receptor CXCR2 was expressed at greater levels by MF splenic littoral cells that line the sinusoids as well as endothelial cells as compared to normal spleens. We hypothesize that effective treatment of MF would require a combination of drugs that directly deplete the malignant HSC/HPCs as well as dampen the tumor promoting microenvironment. In a previous study, we have found the MDM2 protein, a negative regulator of p53, are highly expressed in PV CD34+ cells from PV patients and can be targeted by nutlin therapy (Lu et al, Blood, 2013). In order to assess if nutlin therapy would deplete MF CD34+ cells, using flow cytometry method, we documented that MDM2 protein expression was significantly increased in MF CD34+ cells as compared to normal CD34+ cells and was higher than that in PV CD34+ cells (Figure 2). This finding suggests that MF malignant HSC/HPC might also be sensitive to the treatment with a nutlin. Furthermore, MF HSC/HPCs reside in the BM and extra medullary sites containing dysfunctional microenvironments which promote the predominance of the malignant stem cells. We assume that BET inhibitors might dampen the MF inflammatory milieu since BET proteins play a central role in regulation of inflammatory responses. BET proteins interact with transcription factors (TF) including NF-KB, P53 and c-Myc, and downregulate the transcriptional activation of numerous cytokines including IL-6 and IL-8. We then hypothesize that treatment with low doses of nutlin combined with JQ1, a BET inhibitor, will target MF malignant cells as well as correct MF HSC tissue specific supportive microenvironments by decreasing pro-inflammatory cytokine generation.Results: 1. Treatment with a combination of low dose RG7112 and JQ1 selectively decreased hematopoietic colony formation by MF CD34+ cells. Exposure to either low dose RG7112 (200nM) or JQ1 (50nM) alone for 2 days increased the degree of MF CD34+ cell apoptosis modestly, while a combination of the 2 drugs significantly induced MF CD34+ cell apoptosis (p<0.05). By contrast, treatment with each drug alone or in combination however did not significant induce apoptosis of normal CD34+ cells. Combination treatment of RG7112 and JQ1 inhibited total number of colonies generated by MF CD34+ cells by 60%, as compared to the treatment with each drug alone (p=0.004 and p=0.03, respectively). Combination treatment with RG7112 and JQ1 did not, however, decrease colony formation by normal HPC. In a small number of cases, combination therapy decreased the numbers of homozygous JAK2V617F+ colonies assayed from MF CD34+ cells (at range from 8% to 20%).2. Both RG7112 and JQ1 treatment decrease the elaboration of MF pro-inflammatory cytokines. Treatment of MF mononuclear cells (MNC) with RG7112 but not JQ1 decreased LCN2 levels in conditioned medium (CM) (at range from 5% to 39%, p=0.012); while treatment with JQ1 at 50nM strongly decreased the levels of IL-8 in MF MNC CM by 50% (p=0.0003).Summary: These data indicate that JQ1 combined with RG7112 might be a potential effective strategy for the treatment of MF. This drug combination will not only selectively target MF HSC/HPCs but also alter the MF HSC promoting microenvironment by inhibiting the elaboration of pro-inflammatory mediators. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Protein Synthesis Rates Regulate Tumor-Initiating Potential and Chemoresistance in Multiple Myeloma

Introduction: Multiple myeloma (MM) remains incurable despite improved remissions with novel agents. Relapse eventually occurs in the form of drug-resistant disease that carries a dismal prognosis. Relapse is dependent on stem cell functions such as chemoresistance and self-renewal for the maintenance of clonogenic potential over time. A better understanding of the drivers of these functions may lead to novel therapies for relapsed disease. Although MM is prototypically characterized by antibody secreting plasma cells, disease progression has been correlated with loss of protein production and clonal dominance of undifferentiated plasma cell precursors. We hypothesized that low protein translation rates in MM may enhance key stem cell functions, thus exposing a novel therapeutic opportunity for patients with advanced disease.Methods and Results: MM cell lines were treated with cycloheximide (CHX), a small molecule inhibitor of ribosomal elongation, to determine whether modulating protein translation rates directly impacts sensitivity of MM cells to chemotherapy. At sub-lethal doses, CHX inhibited protein translation by 35S-methionine incorporation in MM cells (NCI-H929 and MM1.S), but increased the relative IC50 of cells to bortezomib, dexamethasone, and melphalan in cell viability assays (range 1.5-5-fold, p-value <0.05). EIF5, a translation initiation complex subunit that can also inhibit translation, was upregulated in response of cells to bortezomib as measured by qRT-PCR (100-fold in NCI-H929, 30-fold in MM1.S). Consistently, forced over-expression of EIF5 in these cell lines significantly inhibited protein translation (range 2.5-3-fold, p-value <0.05), and, similar to CHX, also increased the relative IC50 for multiple chemotherapeutic agents (range 1.8-3-fold, p<0.05). We also found EIF5 to be enriched in progenitor-like, CD138 negative cells in MM cell lines (2.1-2.6-fold, p<0.05) and patient samples (1.5-7.3-fold), which correlated with decreased protein translation rates in these cells (7.4-13.6-fold, p<0.05). Modulation of protein translation rates can influence stem cell functions in normal hematopoiesis, and so we next tested the impact of inhibiting protein translation on MM tumor stem cell functions. In MM cell lines, over-expression of EIF5, or treatment with CHX, significantly enhanced clonogenic growth in vitro as determined in methylcellulose-based colony forming cell assays (2.1-4-fold, p<0.05). Moreover, in a limiting dilution assay, EIF5 expression enhanced tumor initiating cell (TIC) frequency in MM1.S cells that were subcutaneously implanted into immunodeficient mice (TIC frequency of 1/4358 for control vs. 1/262for EIF5 expressing cells; p-value = 0.000003). To explore whether enhancing protein translation has the opposite effect, thus being of possible therapeutic value, we exposed cells to L-leucine, an amino acid that can stimulate protein translation rates when given in excess. L-leucine treatment of MM cell lines significantly decreased colony formation as a single agent (1.5-2-fold, p<0.05).Conclusion: We have shown that protein translation rates are linked to chemoresistance and stem cell functions in MM. Modulating protein translation is a novel therapeutic opportunity for patients with relapsed disease. Conversely, therapies that inhibit protein synthesis rates may paradoxically lead to poor outcomes. DisclosuresNo relevant conflicts of interest to declare.

Silencing of Carboxypeptidase E expression inhibits proliferation and invasion of Panc-1 pancreatic cancer cells

Background: Pancreatic cancer is one of the leading cause of cancer-related death globally. The molecular basis of this disease is complex and not fully understood. Previous studies have indicated that carboxypeptidase E (CPE) plays a role in promoting tumorigenesis in many cancer types. Here we have investigated the effect of carboxypeptidase E (CPE), including its isoform, in regulating the proliferation, migration and invasion of Panc-1 cells, a pancreatic cell line. Methods: Panc-1 cells were transfected with CPE siRNA which targets both CPE-wild type and its isoform, or scrambled siRNA, for 24 h and then assayed for proliferation by the MTT and colony formation assays, and migration and invasion by wound healing and matrigel assays, respectively. Results: CPE siRNA treatment of Panc-1 cells down-regulated the expression of CPE mRNA by 94.8%. Silencing of CPE mRNA expression resulted in a significant decrease in proliferation as revealed by the MTT assay and a 62.8% decrease in colony formation. Western blot analysis of expression of Cyclin D1 in Panc-1 cells treated with CPE siRNA showed a decrease of 32.5% compared to scr siRNA treated cells, indicating that CPE regulates proliferation through modulating this cell cycle protein. Additionally, suppression of CPE expression in Panc-1 cells significantly decreased migration and invasion. Conclusions: Our findings indicate that CPE may play an important role in regulating cell proliferation, migration and invasion to promote pancreatic cancer tumorigenesis.

ITGB6-Knockout Suppresses Cholangiocarcinoma Cell Migration and Invasion with Declining PODXL2 Expression.

Intrahepatic cholangiocarcinoma (iCCA) is a heterogeneous bile duct cancer with a poor prognosis. Integrin αvβ6 (β6) has been shown to be upregulated in iCCA and is associated with its subclassification and clinicopathological features. In the present study, two ITGB6-knockout HuCCT1 CCA cell lines (ITGB6-ko cells) were established using the clustered regulatory interspaced short palindromic repeats (CRISPR), an associated nuclease 9 (Cas9) system, and single-cell cloning. RNA sequencing analysis, real-time polymerase chain reaction (PCR), and immunofluorescent methods were applied to explore possible downstream factors. ITGB6-ko cells showed significantly decreased expression of integrin β6 on flow cytometric analysis. Both cell lines exhibited significant inhibition of cell migration and invasion, decreased wound-healing capability, decreased colony formation ability, and cell cycle dysregulation. RNA sequencing and real-time PCR analysis revealed a remarkable decrease in podocalyxin-like protein 2 (PODXL2) expression in ITGB6-ko cells. Colocalization of PODXL2 and integrin β6 was also observed. S100 calcium-binding protein P and mucin 1, which are associated with CCA subclassification, were downregulated in ITGB6-ko cells. These results describe the successful generation of ITGB6-ko CCA cell clones with decreased migration and invasion and downregulation of PODXL2, suggesting the utility of integrin β6 as a possible therapeutic target or diagnostic marker candidate.

Trilostane, a 3β-hydroxysteroid dehydrogenase inhibitor, suppresses growth of hepatocellular carcinoma and enhances anti-cancer effects of sorafenib.

BackgroundHuman 3β-hydroxysteroid dehydrogenase type 1 (HSD3B1) is an enzyme associated with steroidogenesis, however its' role in hepatocellular carcinoma (HCC) biology is unknown. Trilostane is an inhibitor of HSD3B1 and has been tested as a treatment for patients with breast cancer but has not been studied in patients with HCC. Methods and ResultsThe expression of HSD3B1 in HCC tumors in 57 patients were examined. A total of 44 out of 57 tumors (77.2%) showed increased HSD3B1 expression. The increased HSD3B1 in tumors was significantly associated with advanced HCC. In vitro, the knockdown of HSD3B1 expression in Mahlavu HCC cells by a short hairpin RNA (shRNA) led to significant decreases in colony formation and cell migration. The suppression of clonogenicity in the HSD3B1-knockdown HCC cells was reversed by testosterone and 17β-estradiol. Trilostane-mediated inhibition of HSD3B1 in different HCC cells also caused significant inhibition of clonogenicity and cell migration. In subcutaneous HCC Mahlavu xenografts, trilostane (30 or 60 mg/kg, intraperitoneal injection) significantly inhibited tumor growth in a dose-dependent manner. Furthermore, the combination of trilostane and sorafenib significantly enhanced the inhibition of clonogenicity and xenograft growth, surpassing the effects of each drug used alone, with no documented additional toxicity to animals. HSD3B1 blockade was found to suppress the phosphorylation of extracellular signal-regulated kinase (ERK). The decreased ERK phosphorylation was reversed by testosterone or 17b-estradiol. ConclusionsTrilostane significantly inhibited the growth of HCC by inhibiting HSD3B1 function and augmenting the efficacy of sorafenib.

Blocking hsa_circ_0006168 suppresses cell proliferation and motility of human glioblastoma cells by regulating hsa_circ_0006168/miR-628-5p/IGF1R ceRNA axis

ABSTRACT Background hsa_circ_0006168 is an oncogenic circular RNA in esophageal cancer. However, its role remains unclarified in tumor progression of gliomas, especially in glioblastoma (GBM). Methods Cell counting kit-8 assay, transwell assays, western blotting, and xenograft experiment, as well as colony formation assay and flow cytometry were performed to measure cell proliferation and motility. Expression of hsa_circ_0006168, microRNA (miR)-628-3p, insulin‑like growth factor 1 receptor (IGF1R), and Ras/extracellular signal regulated kinases (Erk)-related proteins were determined by quantitative real-time polymerase chain reaction and western blotting. The physical interaction was confirmed by dual-luciferase reporter assay and RNA pull-down assay. Results hsa_circ_0006168 and IGF1R were upregulated, and miR-628-5p was downregulated in human GBM tissues and cells. Functionally, blocking hsa_circ_0006168 and overexpressing miR-628-5p suppressed cell proliferation, migration, invasion, and expression of Vimentin and Snail (mesenchymal markers) in A172 and LN229 cells, accompanied with increased E-cadherin (epithelial marker), decreased colony formation, and promoted apoptosis rate. Silencing miR-628-5p counteracted the suppression of hsa_circ_0006168 deficiency on these behaviors, and restoring IGF1R blocked miR-628-5p-mediated inhibition as well. More importantly, hsa_circ_0006168 knockdown could delay xenograft tumor growth in vivo and lower Ras and phosphorylated Erk1/2 expression in vitro and in vivo. Mechanically, hsa_circ_0006168 targeted and sponged miR-628-5p, and IFG1R was a novel target for miR-628-5p. Inhibiting miR-628-5p could abrogate in vitro role of hsa_circ_0006168 knockdown, and similarly IGF1R upregulation counteracted miR-628-5p role. Conclusion Silencing hsa_circ_0006168 might suppress GBM proliferation and motility via serving as competitive endogenous RNA for miR-628-5p and regulating IGF1R/Ras/Erk pathway.

Exploiting the anticancer effects of a nitrogen bisphosphonate nanomedicine for glioblastoma multiforme

Glioblastoma multiforme (GBM) is an incurable aggressive brain cancer in which current treatment strategies have demonstrated limited survival benefit. In recent years, nitrogen-containing bisphosphonates (N-BPs) have demonstrated direct anticancer effects in a number of tumour types including GBM. In this study, a nano-formulation with the RALA peptide was used to complex the N-BP, alendronate (ALN) into nanoparticles (NPs) < 200 nm for optimal endocytic uptake. Fluorescently labelled AlexaFluor®647 Risedronate was used as a fluorescent analogue to visualise the intracellular delivery of N-BPs in both LN229 and T98G GBM cells. RALA NPs were effectively taken up by GBM where a dose-dependent response was evidenced with potentiation factors of 14.96 and 13.4 relative to ALN alone after 72 h in LN229 and T98G cells, respectively. Furthermore, RALA/ALN NPs at the IC50, significantly decreased colony formation, induced apoptosis and slowed spheroid growth in vitro. In addition, H-Ras membrane localisation was significantly reduced in the RALA/ALN groups compared to ALN or controls, indicative of prenylation inhibition. The RALA/ALN NPs were lyophilised to enhance stability without compromising the physiochemical properties necessary for functionality, highlighting the suitability of the NPs for scale-up and in vivo application. Collectively, these data show the significant potential of RALA/ALN NPs as novel therapeutics in the treatment of GBM.

GH Is a Component of SASP in Aging Tissue

Deficient GH signaling results in lifespan extension in murine and human models, while patients with uncontrolled acromegaly and transgenic mice overexpressing GH have a shorter lifespan. Colon polyp development increases with age, and also with GH excess in acromegaly. Aging is characterized by senescent cell accumulation with p53/p21 or p16 upregulation as well as cell cycle arrest and expression of a senescence-associated secretory phenotype (SASP). Senescence is reinforced by the SASP, which comprises pro-inflammatory cytokines, chemokines, growth modulators, angiogenic factors, and matrix metalloproteinases. SASP contributes to pro-aging phenotypes, and depletion or removal of senescent cells increases lifespan by partially protecting from age-related pathologies. Senescence, triggered by genotoxic insult with DNA damage, can be reversed by p53 inactivation; senescent cells with low p53 and unrepaired DNA damage can then re-enter the cell cycle (Beausejour et al, EMBO 2003), potentially resulting in chromosomal instability. We showed GH induction in non-pituitary senescent cells leading to suppressed p53/p21 (Chesnokova PNAS 2013). We now show that, in senescent human colon cells (hNCC) and in 3-dimensional human intestinal organoids, non-pituitary GH (npGH) is a component of SASP. In response to DNA damage, npGH is expressed and secreted locally as measured by RT-PCR, WB, and ELISA. High autocrine/paracrine GH further exacerbate DNA damage and reverses senescent in colon cells, enabling them to re-enter the cell cycle, as evidenced by p53 downregulation and increased Ki67 expression. Senescent colon cells expressing high intracellular GH form colonies in soft agar, indicative of cell transformation and proliferation, while GH deletion by shRNA results in Ki67 downregulation and decreased colony formation and size. The SASP protein CXCL1, a chemo-attractant that also functions to eliminate senescent cells, dose-dependently activates GH in hNCC and in intestinal organoids. GH, in turn, suppresses CXCL1 expression. Consistent with these findings, colon CXCL1 was induced in GHRKO mice, and also in hNCC with abrogated GH signaling by shRNA. Taken together with our finding that GH accumulates in aging normal human colon tissue and co-localizes in cells expressing senescence-associated β-galactosidase, these results suggest that GH, as a SASP component, initiates senescent cell proliferation and transformation. By inhibiting CXCL1, GH also functions to attenuate immune-mediated senescent cell elimination that protects aging tissue from deleterious effects of SASP. These mechanisms may underly an initial step in age-associated epithelial polyp development.

Activation of plasmacytoid dendritic cells promotes AML-cell fratricide

Acute myeloid leukemia (AML) is characterized by the proliferation of immature myeloid blasts and a suppressed immune state. Interferons have been previously shown to aid in the clearance of AML cells. Type I interferons are produced primarily by plasmacytoid dendritic cells (pDCs). However, these cells exist in a quiescent state in AML. Because pDCs express TLR 7–9, we hypothesized that the TLR7/8 agonist R848 would be able to reprogram them toward a more active, IFN-producing phenotype. Consistent with this notion, we found that R848-treated pDCs from patients produced significantly elevated levels of IFNβ. In addition, they showed increased expression of the immune-stimulatory receptor CD40. We next tested whether IFNβ would influence antibody-mediated fratricide among AML cells, as our recent work showed that AML cells could undergo cell-to cell killing in the presence of the CD38 antibody daratumumab. We found that IFNβ treatment led to a significant, IRF9-dependent increase in CD38 expression and a subsequent increase in daratumumab-mediated cytotoxicity and decreased colony formation. These findings suggest that the tolerogenic phenotype of pDCs in AML can be reversed, and also demonstrate a possible means of enhancing endogenous Type I IFN production that would promote daratumumab-mediated clearance of AML cells.

Metformin impairs cisplatin resistance effects in A549 lung cancer cells through mTOR signaling and other metabolic pathways.

Lung cancer is the leading cause of cancer-associated death worldwide and exhibits intrinsic and acquired therapeutic resistance to cisplatin (CIS). The present study investigated the role of mTOR signaling and other signaling pathways after metformin (MET) treatment in control and cisplatin-resistant A549 cells, mapping pathways and possible targets involved in CIS sensitivity. MTT, flow cytometry, clonogenic assay, western blotting, proteomic analysis using the Stable Isotope Labeling by Amino acids in Cell culture (SILAC) approach and reverse transcription-quantitative PCR were performed. The results revealed that CIS treatment induced mTOR signaling pathway overactivation, and the mTOR status was restored by MET. MET and the mTOR inhibitor rapamycin (RAPA) decreased the viability in control and resistant cells, and decreased the cell size increase induced by CIS. In control cells, MET and RAPA decreased colony formation after 72 h and decreased IC50 values, potentiating the effects of CIS. Proteomics analysis revealed important pathways regulated by MET, including transcription, RNA processing and IL-12-mediated signaling. In CIS-resistant cells, MET regulated the apoptotic process, oxidative stress and G2/M transition. Annexin 4 (ANXA4) and superoxide dismutase 2 (SOD2), involved in apoptosis and oxidative stress, respectively, were chosen to validate the SILAC analysis and may represent potential therapeutic targets for lung cancer treatment. In conclusion, the chemosensitizing and antiproliferative effects of MET were associated with mTOR signaling and with potential novel targets, such as ANXA4 and SOD2, in human lung cancer cells.

The Effect of EZH2 Inhibition through DZNep on Epithelial-Mesenchymal Transition Mechanism.

Although the molecular pathogenesis of hepatocellular carcinoma (HCC) is uncertain, it is known that the epithelial-mesenchymal transition (EMT) mechanism and epigenetic changes have an important role. This study was focused on evaluating the relationship of 3-Deazaneplanocin A (DZNep) with the EMT mechanism, which is a histone methyltransferase inhibitor on HCC and is also known as an enhancer of zeste homolog 2 (EZH2) inhibitor. Cell viability of HepG2 cells (HCC cell line) assessed for DZNep over 72 hours with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Additionally, colony-forming assay, apoptosis assay, RNA isolation, cDNA synthesis, and real-time PCR (RT-PCR) were performed to see the effect of DZNep on HepG2 cells. DZNep reduced cell proliferation for 72 hours, also significantly reduced colony formation in addition it increased the total apoptosis. DZNep on EZH2, E-cadherin, N-cadherin, and Vimentin (Vim) gene expressions was given different results by either decreasing or increasing the expressions. In this study, we observed a positive effect of DZNep on apoptosis and TIMP3 expression level and decreased colony formation. However, it gave complicated results with the level of gene expression E-cadherin and TIMP2, increase the level of Vim and MMP2 expression. Therefore, we think that further studies are necessary to clarify the role of DZNep.

Präklinische Untersuchungen von Wechselwirkungen zwischen Mistel und Radio- oder Chemotherapie auf pädiatrische Tumorzellen

Hintergrund: Mistelanwendungen werden als komplementäre Therapien häufig in der pädiatrischen Onkologie zusammen mit einer Radio- oder Chemotherapie verabreicht. Wechselwirkungen bei simultaner Applikation sind gerade in der Pädiatrie von großer Bedeutung, sie sind allerdings nach wie vor unzureichend untersucht. Material und Methoden: Zytotoxische Effekte des Mistelextraktes abnobaVISCUM Fraxini (aVF) auf LAN-1 Neuroblastomzellen und deren Etoposid- bzw. Cisplatin-resistente Subzelllinien wurden mittels Viabilitätstest untersucht, sowie mögliche Synergieeffekte zwischen aVF und den Chemotherapeutika durch die Softwareprodukte Combenefit und CompuSyn analysiert. Effekte einer Kombinationstherapie aus aVF und Bestrahlung auf SH-SY5Y Zellen wurden mittels Koloniebildungstest untersucht und Auswirkungen auf die Reparatureffizienz strahleninduzierter Doppelstrangbrüche mit Hilfe durchflusszytometrischer Quantifizierungen von γ-H2AX-Foci nach PI/FITC Doppelfärbung analysiert. Ergebnisse: Die Chemotherapie-resistenten LAN-1 Subzelllinien erwiesen sich als resistenter gegenüber der Mistelbehandlung als die Ursprungszelllinie. Auf Basis vier verschiedener Referenz­modelle konnten vor allem synergistisch/additive Effekte zwischen aVF und den Zytostatika Etoposid und Cisplatin berechnet werden. Die Kombination aus Mistelbehandlung und Bestrahlung führte zu einer Verringerung der Koloniebildung und zu einer Verzögerung der Reparaturgeschwindigkeit von strahleninduzierten Doppelstrangbrüchen. Schlussfolgerung: Die präklinischen Daten könnten darauf hinweisen, dass die Verwendung des Mistelextraktes, aVF, eine unterstützende Wirkung auf Radio- und Chemotherapien hat.

Bortezomib suppresses self-renewal and leukemogenesis of leukemia stem cell by NF-ĸB-dependent inhibition of CDK6 in MLL-rearranged myeloid leukemia.

Acute myeloid leukaemia (AML) with chromosomal rearrangements involving the H3K4 methyltransferase mixed‐lineage leukaemia (MLL) is an aggressive subtype with low overall survival. Bortezomib (Bort) is first applied in multiple myeloma. However, whether bort possesses anti‐self‐renewal and leukemogenesis of leukaemia stem cell (LSC) in AML with MLL rearrangements is still unclear. Here, we found that bort suppressed cell proliferation and decreased colony formation in human and murine leukaemic blasts. Besides, bort reduced the frequency and function of LSC, inhibited the progression, and extended the overall survival in MLL‐AF9 (MF9) ‐transformed leukaemic mice. Furthermore, bort decreased the percentage of human LSC (CD34+CD38‐) cells and extended the overall survival in AML blasts‐xenografted NOD/SCID‐IL2Rγ (NSG) mice. Mechanistically, cyclin dependent kinase 6 (CDK6) was identified as a bort target by RNA sequencing. Bort reduced the expressions of CDK6 by inhibiting NF ĸB recruitment to the promoter of CDK6, leading to the abolishment of NF ĸB DNA‐binding activity for CDK6 promoter. Overexpression of CDK6 partially rescued bort‐induced anti‐leukemogenesis. Most importantly, bort had little side‐effect against the normal haematological stem and progenitor cell (HSPC) and did not affect CDK6 expression in normal HSPC. In conclusion, our results suggest that bort selectively targets LSC in MLL rearrangements. Bort might be a prospective drug for AML patients bearing MLL rearrangements.