Anchorage-independent Colony Formation Research Articles

Modification of ERα by UFM1 Increases Its Stability and Transactivity for Breast Cancer Development.

The post-translational modification (e.g., phosphorylation) of estrogen receptor α (ERα) plays a role in controlling the expression and subcellular localization of ERα as well as its sensitivity to hormone response. Here, we show that ERα is also modified by UFM1 and this modification (ufmylation) plays a crucial role in promoting the stability and transactivity of ERα, which in turn promotes breast cancer development. The elevation of ufmylation via the knockdown of UFSP2 (the UFM1-deconjugating enzyme in humans) dramatically increases ERα stability by inhibiting ubiquitination. In contrast, ERα stability is decreased by the prevention of ufmylation via the silencing of UBA5 (the UFM1-activating E1 enzyme). Lys171 and Lys180 of ERα were identified as the major UFM1 acceptor sites, and the replacement of both Lys residues by Arg (2KR mutation) markedly reduced ERα stability. Moreover, the 2KR mutation abrogated the 17β-estradiol-induced transactivity of ERα and the expression of its downstream target genes, including pS2, cyclin D1, and c-Myc; this indicates that ERα ufmylation is required for its transactivation function. In addition, the 2KR mutation prevented anchorage-independent colony formation by MCF7 cells. Most notably, the expression of UFM1 and its conjugating machinery (i.e., UBA5, UFC1, UFL1, and UFBP1) were dramatically upregulated in ERα-positive breast cancer cell lines and tissues. Collectively, these findings implicate a critical role attributed to ERα ufmylation in breast cancer development by ameliorating its stability and transactivity.

IDH1 Mutation Induces HIF-1α and Confers Angiogenic Properties in Chondrosarcoma JJ012 Cells.

Chondrosarcoma is a group of primary bone cancers that arise from transformed cells of chondrocytic lineage. Tumor recurrence and metastasis are devastating for patients with chondrosarcoma since there are no effective treatment options. IDH mutations occur in over 50% of tumors from patients with conventional or dedifferentiated chondrosarcomas and represent an attractive target for therapy. However, their role in the pathogenesis of chondrosarcoma remains largely unknown. In this study, we sought to determine the association of IDH mutation and HIF-1α in chondrosarcoma. We used the chondrosarcoma JJ012 cell line and its derived CRISPR/Cas9 mutant IDH1 (IDH1mut) knockout (KO) cells. RNA-Seq data analysis revealed downregulation of several HIF-1α target genes upon loss of IDH1mut. This was associated with reduced HIF-1α levels in the IDH1mut KO cells and tumors. Loss of IDH1mut also attenuated the expression of angiogenic markers in tumor tissues and abrogated the angiogenic capacity of JJ012 cells. Moreover, we observed that exogenous expression of HIF-1α significantly promoted anchorage-independent colony-formation by IDH1mut KO cells. These results suggest IDH1 mutation confers angiogenic and tumorigenic properties of JJ012 cells by inducing HIF-1α. Thus, the HIF pathway represents a promising candidate for combinatorial regimens to target IDH1 mutated chondrosarcomas.

Anti-Proliferative and Pro-Apoptotic Effects Of Dipsacus Asperoides in a Cellular Model for Triple-Negative Breast Cancer

Background: Triple negative breast cancer (TNBC) lacks expressions of estrogen receptor-α (ER-α), progesterone receptor (PR) and amplified human epidermal growth factor receptor-2 (HER-2). Current treatment for TNBC includes anthracyclin, taxol and cisplatin-based conventional chemotherapy and survival pathway PARP, PI3K, AKT and mTOR selective targeted therapy. These treatments exhibit dose-limiting systemic toxicity and presence of drug resistant cancer stem cells, which highlight the need for identification of efficacious testable alternatives that are not toxic to non-tumorigenic cells. Dipsacus asperoides (DA) is a Chinese nutritional herb and its root represents a common ingredient in Chinese herbal formulations used in women for estrogen related health issues, osteoporosis and breast diseases. This study aims to investigate the growth inhibitory effects of DA, and to detect mechanisms for its efficacy. Methods: Human mammary carcinoma derived triple negative MDA-MB-231 cell line represented the TNBC model. Non-fractionated aqueous extract from DA represented the test agent. Anchorage dependent growth, anchorage independent (AI) colony formation and cell cycle progression quantified growth inhibition. Western blot-based analysis for inhibition of RAS, PI3K and AKT and RB signaling identified mechanistic leads. Results: Treatment with DA induced a dose dependent cytostatic growth arrest (IC50:15 µg/ml; IC90: 30 µg/ml), reduced AI growth and inhibited cell cycle progression via G2/M arrest. DA affected the RAS, PI3K, AKT and RB signaling pathways, and functioned as a natural inhibitor of cyclin dependent kinase 4/6. Cellular apoptosis paralleled increase in pro-apoptotic Caspase 3/7 activity. Conclusion: These results demonstrate that DA inhibited growth, affected cell cycle progression, induced apoptosis and inhibited cancer cell survival pathways. This study validates a mechanism-based approach to identifying testable substitutes for secondary prevention/therapy of TNBC.

Co-upregulation of miR-31 and its host gene lncRNA MIR31HG in oral squamous cell carcinoma

Background/purposeSeveral long non-coding RNAs (lncRNAs) harbor miRNA in their genome. MIR31HG harbors miR-31 in its intron and it is speculated that they are co-expressed in tumors. This study addressed whether frequent miR-31 and MIR31HG co-upregulation occurred in oral squamous cell carcinoma (OSCC) and its clinical implications. Materials and methodsMicroarray was performed to retrieve dis-regulated lncRNAs from tissue sample. The ectopic gene expression was carried out to specify the phenotypic influences of selected lncRNA screened from bioinformatic algorithms. The expression of miR-31 and MIR31HG in tissues or scrapped samples was analyzed using qRT-PCR. The implications of gene expression as related to metastasis or survival were further dissected. ResultsMicroarray identified disrupted transcripts including MIR31HG and other 152 lncRNAs aberrantly expressed in OSCC tissues. In silico algorithms annotated an eminent involvement of aberrant transcripts in the regulation of cell cycle, extracellular modulation, adhesion, and wound healing. The enhancement of proliferation, wound healing, invasion and anchorage-independent colony formation mediated by MIR31HG was ascertained by ectopic expression in OECM1 cells. Besides, co-upregulation of miR-31 and MIR31HG was conspicuous in OSCC tissues. High expression of miR-31 and MIR31HG designated a trend of worse OSCC prognosis. Interestingly, high MIR31HG expression defined a very poor survival in stage IV diseases. By contrast, high miR-31 expression predicted nodal metastasis in stage I–III diseases. ConclusionAssessment of miR-31 and MIR31HG expression in OSCC may enable the prognostic prediction. The candidate lncRNAs isolated from this work can be further validated as crucial factors contributing to OSCC pathogenesis.

Anticancer effects of veratramine via the phosphatidylinositol-3-kinase/serine-threonine kinase/mechanistic target of rapamycin and its downstream signaling pathways in human glioblastoma cell lines

AimsAntitumor effects of veratramine in prostate and liver cancers has been investigated, but it is still unclear whether veratramine can be used as an effective therapeutic agent for glioma. The aim of this study was to evaluate the potential pharmacological mechanism of veratramine in glioma. Main methodsUsing four types of human glioblastoma cell lines, including A172, HS-683, T98G, and U-373-MG the dose-dependent antitumor effect of veratramine was evaluated. The cytotoxicity and cell proliferation were examined by CCK-8, and cell proliferation was further confirmed by anchorage-independent colony formation assay. The cell cycle distribution and apoptotic rate was assessed by flow cytometry, and apoptosis was further evaluated by apoptosis assay. The migration and invasiveness capacity were analyzed by using transwell. Protein and mRNA levels of related factors were determined by western blotting and RT-qPCR, respectively. Key findingsVeratramine markedly induced apoptosis, suppressed the cell proliferation via the cell cycle G0/G1 phase arrest, and reduced the capacity for the migration and invasion in human glioblastoma multiforme cell lines. Moreover, veratramine was sufficient to affect the phosphatidylinositol-3-kinase/serine-threonine kinase/mechanistic target of rapamycin signaling pathway and its downstream Mdm2/p53/p21 pathway in human glioblastoma cell lines. SignificanceAntitumor effects of veratramine in suppression of glioma progression was mediated by the regulation of PI3K/Akt/mTOR and Mdm2/p53/p21 signaling pathway.

Abemaciclib is synergistic with doxorubicin in osteosarcoma pre-clinical models via inhibition of CDK4/6-Cyclin D-Rb pathway.

Osteosarcoma is a prevalent type of bone tumor in children and adolescents, with limited treatment and poor prognosis. Abemaciclib, an inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6), is approved for the treatment of advanced breast cancer as single agent therapy and is currently under investigation in clinical trials for the treatment of several solid tumors. The efficacy of abemaciclib was determined using osteosarcoma cellular assays and xenograft mouse model. The combination studies were performed based on the Chou-Talalay method. Immunoblotting analysis was performed to determine the underlying mechanisms of abemaciclib in osteosarcoma cell lines. Abemaciclib potently inhibits growth, anchorage-independent colony formation and survival of a panel of osteosarcoma cell lines, with IC50 range from 90 nM to >20 μM. In addition, the combination of abemaciclib and doxorubicin is synergistic and antagonistic in abemaciclib-sensitive (IC50<1 μM) and abemaciclib-resistant (IC50 >1 μM), respectively. Abemaciclib inhibits tumor formation and growth in a dose-dependent manner without causing significant drug toxicity in mice. The combination of abemaciclib and doxorubicin results in much greater efficacy than doxorubicin alone in inhibiting tumor growth throughout the whole treatment duration. Abemaciclib acts on osteosarcoma via suppressing CDK4/6-Cyclin D-Rb pathway. Our pre-clinical evidence provides a rationale of initializing clinical trial of investigating the efficacy of abemaciclib in combination with doxorubicin in osteosarcoma patients. Our work also highlights the therapeutic value of CDK4/6 inhibition in osteosarcoma with proper function of Rb.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Growth Inhibitory Efficacy of Tabebuia Avellanedae in a Model for Triple Negative Breast Cancer

Background: Tabebuia avellanedae (TA) is a tree that is indigenous to the Amazon rainforest. The experiments in the present study were designed to examine the inhibitory effects of TA, and to identify mechanistic targets for its efficacy in the estrogen-α receptor (ER-α), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2) negative MDA-MB-231 model for triple negative breast cancer (TNBC). Methods: Non-fractionated aqueous extract from the inner bark of TA was used in the experiments. Anchorage dependent growth, anchorage independent (AI) colony formation, cell cycle progression, and expressions of relevant regulatory proteins represented quantitative end points. Results: Long-term treatment for 21 days with the maximum cytostatic concentration of 2.5% TA resulted in a 90% inhibition (P=0.014) in AI colony number. Short-term treatment for 2 days with 1.0% TA (IC50) resulted in about a 1.3 fold increase (P=0.014) in G1: S+G2/M ratio, about a 1.48 fold increase (P=0.010) in the sub G0 (apoptotic) cells and about a 3.2 fold increase (P=0.014) in the pro-apoptotic caspase 3/7 activity. Mechanistically, the short-term treatment with 2.5% TA decreased Cyclin D1 expression by about 83.3%, and pRB expression by about 73.3%. Conclusion: TNBC represents an aggressive cancer notable for its resistance to conventional and targeted therapy. Non-toxic natural substances may represent testable alternatives. This study identifies potential mechanistic leads for TA as a novel naturally occurring testable alternative for secondary prevention/therapy of TNBC, and validates a novel mechanistic approach to evaluate efficacious non-toxic phytochemicals and herbs as testable alternatives against therapy resistant breast cancer.

Stemness-Suppressive Effect of Bibenzyl from Dendrobium ellipsophyllum in Human Lung Cancer Stem-Like Cells.

Cancer stem-like cells (CSCs) are key mediators driving tumor initiation, metastasis, therapeutic failure, and subsequent cancer relapse. Thus, targeting CSCs has recently emerged as a potential strategy to improve chemotherapy. In this study, the anticancer activity and stemness-regulating capacity of 4,5,4′-trihydroxy-3,3′-dimethoxybibenzyl (TDB), a bibenzyl extracted from Dendrobium ellipsophyllum, are revealed in CSCs of various human lung cancer cells. Culture with TDB (5–10 μM) strongly abolished tumor-initiating cells in lung cancer H460, H23, and A549 cells in both anchorage-dependent and anchorage-independent colony formation assays. Through the 3D single-spheroid formation model, attenuation of self-renewal capacity was observed in CSC-enriched populations treated with 1–10 μM TDB for 7 days. Flow cytometry analysis confirmed the attenuation of %cell overexpressing CD133, a CSC biomarker, in TDB-treated lung cancer spheroids. TDB at 5–10 μM remarkably suppressed regulatory signals of p-Akt/Akt, p-GSK3β/GSK3β, and β-catenin corresponding to the downregulated mRNA level of stemness transcription factors including Nanog, Oct4, and Sox2. Moreover, the antiapoptosis Bcl-2 and Mcl-1 proteins, which are downstream molecules of Akt signaling, were evidently decreased in CSC-enriched spheroids after culture with TDB (1–10 μM) for 24 h. Interestingly, the diminution of Akt expression by specific siAkt effectively reversed suppressive activity of TDB targeting on the CSC phenotype in human lung cancer cells. These findings provide promising evidence of the inhibitory effect of TDB against lung CSCs via suppression of Akt/GSK3β/β-catenin cascade and related proteins, which would facilitate the development of this bibenzyl natural compound as a novel CSC-targeted therapeutic approach for lung cancer treatment.

Physciosporin suppresses mitochondrial respiration, aerobic glycolysis, and tumorigenesis in breast cancer

BackgroundPhysciosporin (PHY) is one of the potent anticancer lichen compound. Recently, PHY was shown to suppress colorectal cancer cell proliferation, motility, and tumorigenesis through novel mechanisms of action. PurposeWe investigated the effects of PHY on energy metabolism and tumorigenicity of the human breast cancer (BC) cells MCF-7 (estrogen and progesterone positive BC) and MDA-MB-231 (triple negative BC). MethodsThe anticancer effect of PHY on cell viability, motility, cancer metabolism and tumorigenicity was evaluated by MTT assay, migration assay, clonogenic assay, anchorage-independent colony formation assay, glycolytic and mitochondrial metabolism analysis, qRT-PCR, flow cytometric analysis, Western blotting, immunohistochemistry in vitro; and by tumorigenicity study with orthotopic breast cancer xenograft model in vivo. ResultsPHY markedly inhibited BC cell viability. Cell-cycle profiling and Annexin V–FITC/PI double staining showed that a toxic dosage of PHY triggered apoptosis in BC cell lines by regulating the B-cell lymphoma-2 (Bcl-2) family proteins and the activity of caspase pathway. At non-toxic concentrations, PHY potently decreased migration, proliferation, and tumorigenesis of BC cells in vitro. Metabolic studies revealed that PHY treatment significantly reduced the bioenergetic profile by decreasing respiration, ATP production, and glycolysis capacity. In addition, PHY significantly altered the levels of mitochondrial (PGC-1α) and glycolysis (GLUT1, HK2 and PKM2) markers, and downregulated transcriptional regulators involved in cancer cell metabolism, including β-catenin, c-Myc, HIF-1α, and NF-κB. An orthotopic implantation mouse model of BC confirmed that PHY treatment suppressed BC growth in vivo and target genes were consistently suppressed in tumor specimens. ConclusionThe findings from our in vitro as well as in vivo studies exhibit that PHY suppresses energy metabolism as well as tumorigenesis in BC. Especially, PHY represents a promising therapeutic effect against hormone-insensitive BC (triple negative) by targeting energy metabolism.

The transcription factor RUNX2 fuels YAP1 signaling and gastric cancer tumorigenesis

Despite considerable efforts in the detection and treatment of gastric cancer (GC), the underlying mechanism of the progression of GC remains unknown. Our previous work has demonstrated the remarkable role of Runt‐related transcription factor 2 (RUNX2), in fueling the invasion and metastasis of GC. The present study aimed to elucidate the role of RUNX2 in tumorigenesis of GC. We assessed Runx2 expression and its clinical significance via bioinformatic analysis of the Cancer Genome Atlas and Gene Expression Omnibus databases. Roles for Runx2 in self‐renewal and tumorigenesis were examined in vitro and in vivo. Further bioinformatic analysis was applied to study the mechanism of GC progression. We found that Runx2 was highly expressed in the early stage of GC and positively correlated with a poor clinical outcome of patients. Runx2 was also significantly correlated with clinicopathological features, such as Hp infection, new neoplastic events, primary therapeutic outcome, ethnicity, race, and tumor stage. Multivariate analysis revealed that together with Runx2, age, cancer status, M stage, and T stage were independent prognostic factors for the outcome of GC patients. RUNX2 overexpression induced increased anchorage‐independent colony formation, sphere formation, and tumorigenesis in GC cells in vitro and in vivo. Mechanistically, bioinformatic analysis indicated that yes1 associated transcriptional regulator (YAP1) might be a downstream target of RUNX2. Specific knockdown of YAP1 reduced the tumor‐initiating ability of GC cells induced by ectopic Runx2 expression. Our findings support the hypothesis that RUNX2 exerts oncogenic properties via YAP1 regulation, highlighting essential roles for RUNX2 and YAP1 in gastric carcinogenesis and suggesting potential therapeutic targets.

Costunolide Induces Apoptosis via the Reactive Oxygen Species and Protein Kinase B Pathway in Oral Cancer Cells.

Oral cancer (OC) has been attracted research attention in recent years as result of its high morbidity and mortality. Costunolide (CTD) possesses potential anticancer and bioactive abilities that have been confirmed in several types of cancers. However, its effects on oral cancer remain unclear. This study investigated the potential anticancer ability and underlying mechanisms of CTD in OC in vivo and in vitro. Cell viability and anchorage-independent colony formation assays were performed to examine the antigrowth effects of CTD on OC cells; assessments for migration and invasion of OC cells were conducted by transwell; Cell cycle and apoptosis were investigated by flow cytometry and verified by immunoblotting. The results revealed that CTD suppressed the proliferation, migration and invasion of oral cancer cells effectively and induced cell cycle arrest and apoptosis; regarding the mechanism, CTD bound to AKT directly by binding assay and repressed AKT activities through kinase assay, which thereby downregulating the downstream of AKT. Furthermore, CTD remarkably promotes the generation of reactive oxygen species by flow cytometry assay, leading to cell apoptosis. Notably, CTD strongly suppresses cell-derived xenograft OC tumor growth in an in vivo mouse model. In conclusion, our results suggested that costunolide might prevent progression of OC and promise to be a novel AKT inhibitor.

Combinatorial Effect of Temozolomide and Naringenin in Human Glioblastoma Multiforme Cell Lines

Glioblastoma multiforme (GBM) is a grade IV, lethal, and the most common type of brain tumor. GBM can acquire resistance to temozolomide (TMZ) recommended for its treatment. Naringenin (NAG), a flavonoid generally found in grapefruit, has antioxidant, anti-proliferative, and anti-inflammatory properties. It has been reported that phytochemicals can reduce resistance and improve the efficacy of a chemo-resistant drug. The combinatorial effect of TMZ and NAG on cell proliferation was evaluated using 3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) assay, and the apoptosis in the U87MG and LN229 GBM cells were evaluated by change in fluorescence intensity. The effect of NAG and TMZ on anchorage-independent single-cell colony formation and cell migration was investigated. NAG and TMZ demonstrated enhanced cytotoxic effects on U87MG and LN229 cell lines. The combination index value being less than one indicated the synergistic action of the two drugs in restricting the growth of the cells. The NAG and TMZ together resulted in higher fluorescence intensity as compared to the alone drug. Further, the study showed a marked reduction in the migration of the cells and the formation of a single cell colony. Supplemental data for this article is available online at https://doi.org/10.1080/01635581.2021.1952438

Abstract 1174: Avenue to personalized targeted therapy for hepatoblastoma

Abstract Introduction: Hepatoblastoma (HB) is the most frequent liver malignancy of childhood with an annual incidence of 1.5 per million and a survival rate less than 50% for high-risk, relapse, and treatment refractory patients. The overall goal of this study is to assess the therapeutic efficacies and anti-cancer mechanisms of novel drugs with HB through a drug sensitivity testing pipeline utilizing multiple novel, clinically relevant preclinical patient-derived cell line (PDCL) and patient-derived xenograft (PDX) models. Methods: A patient derived xenograft (PDX) mouse model was generated with intrahepatic implantation of tumor thrombus from a non-metastatic stage 3 patient who recurred after transplantation. A stable PDCL was grown from the murine tumor, and 60 targeted agents were tested for efficacy with the PDCL by using CellTiter-Glo Luminescent Viability assays. Anti-oncogenic effects of the most efficacious agent, dinaciclib (cyclin-dependent kinase (CDK) inhibitor), were further assayed in vitro with HB cell lines (HepG2, HepT1, Huh6, HB17) with phenotypic assays for cell toxicity (MTT assay), proliferation (CCK-8 assay), and anchorage independent colony formation (soft agar assay). Immunoblotting assays were used to assess changes in drug targets and induction of apoptosis (PARP cleavage) with cell lines. Dinaciclib was also tested in vivo in our orthotopic PDX models of high-risk HB. The drug was administered three times a week by intraperitoneal injection (20 mg/kg, 100 µL) for 3 weeks. Magnetic resonance imaging (MRI) and Alpha-fetoprotein (AFP) ELISA were done to evaluate tumor growth throughout the study as tumor-bearing animals show expression of human serum AFP. Tumor weights and volumes, as well as immunohistochemistry for H&amp;E, Ki-67, and TUNEL, were also used to assess effects of drug in vivo. Results: The agent that showed the lowest IC50 value out of 60 tested agents was the CDK inhibitor, dinaciclib (0.7 µM). Dinaciclib suppressed cell proliferation and viability in a dose-dependent manner in all HB cell lines tested with IC50 values in the low micromolar range (HepG2 3.75 µM; HepT1 0.007 µM; Huh-6 1.42 µM; HB17 0.72 µM). Immunoblotting with dinaciclib-treated cells showed induction of PARP cleavage, indicating apoptosis, along with decreased protein expression of CDK1, 2, 5, and 9. Dinaciclib also showed significant in vivo inhibition of tumor growth compared to placebo (relative tumor volume p = 0.03, tumor weight p = 0.02). Conclusions: Drug screening of a novel HB PDCL identified an agent, dinaciclib, that shows preclinical efficacy with refractory disease. Further in vitro and in vivo validation confirmed that dinaciclib is a promising therapeutic agent for the treatment of HB. Such a personalized medicine pipeline of development of PDCLs and testing them with targeted agents will lead to the identification of new drugs that can be used with patients that do not respond to standard therapies. Citation Format: Rohit Kumar Srivastava, Roma Patel, Richard S. Whitlock, Samuel R. Larson, Andrew Badachhape, Sarah E. Woodfield, Sanjeev A. Vasudevan. Avenue to personalized targeted therapy for hepatoblastoma [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 1174.

AP-2α-Mediated Activation of E2F and EZH2 Drives Melanoma Metastasis.

In melanoma metastasis, the role of the AP-2α transcription factor, which is encoded by TFAP2A, is controversial as some findings have suggested tumor suppressor activity while other studies have shown high TFAP2A expression in node-positive melanoma associated with poor prognosis. Here we demonstrate that AP-2α facilitates melanoma metastasis through transcriptional activation of genes within the E2F pathway including EZH2. A BioID screen found that AP-2α interacts with members of the nucleosome remodeling and deacetylase (NuRD) complex. Loss of AP-2α removed activating chromatin marks in the promoters of EZH2 and other E2F target genes through activation of the NuRD repression complex. In melanoma cells, treatment with tazemetostat, an FDA-approved and highly specific EZH2 inhibitor, substantially reduced anchorage-independent colony formation and demonstrated heritable antimetastatic effects, which were dependent on AP-2α. Single-cell RNA sequencing analysis of a metastatic melanoma mouse model revealed hyperexpansion of Tfap2a High/E2F-activated cell populations in transformed melanoma relative to progenitor melanocyte stem cells. These findings demonstrate that melanoma metastasis is driven by the AP-2α/EZH2 pathway and suggest that AP-2α expression can be used as a biomarker to predict responsiveness to EZH2 inhibitors for the treatment of advanced melanomas. SIGNIFICANCE: AP-2α drives melanoma metastasis by upregulating E2F pathway genes including EZH2 through inhibition of the NuRD repression complex, serving as a biomarker to predict responsiveness to EZH2 inhibitors.

Rhein Suppresses Colorectal Cancer Cell Growth by Inhibiting the mTOR Pathway In Vitro and In Vivo.

Simple SummaryColorectal cancer (CRC) is the fourth most common cancer and the second most common cause of cancer-related deaths globally. Rhein is a natural anthraquinone extract from rhubarb, which exhibits potent anticancer activity in various cancers. In this study, we show that rhein significantly inhibited the growth, migration, and invasion of CRC cells by directly binding to mTOR and inhibiting the mTOR signaling pathway. Rhein promotes mTOR degradation through the ubiquitin–proteasome pathway. In addition, rhein significantly suppressed tumor growth in a xenograft mouse model without obvious toxicity. Our results indicate that rhein is a promising anticancer agent that may be useful for the prevention and treatment of CRC.Colorectal cancer (CRC) is one of the leading causes of mortality and morbidity in the world. Rhein has demonstrated therapeutic effects in various cancer models. However, its effects and underlying mechanisms of action in CRC remain poorly understood. We investigated the potential anticancer activity and underlying mechanisms of rhein in CRC in vitro and in vivo. Cell viability and anchorage-independent colony formation assays were performed to examine the antigrowth effects of rhein on CRC cells. Wound-healing and Transwell assays were conducted to assess cell migration and invasion capacity. Cell cycle and apoptosis were investigated by flow cytometry and verified by immunoblotting. A tissue microarray was used to detect mTOR expression in CRC patient tissues. Gene overexpression and knockdown were done to analyze the function of mTOR in CRC. The anticancer effect of rhein in vivo was assessed in a CRC xenograft mouse model. The results show that rhein significantly inhibited CRC cell growth by inducing S-phase cell cycle arrest and apoptosis. Rhein inhibited CRC cell migration and invasion through the epithelial–mesenchymal transition (EMT) process. mTOR was highly expressed in CRC cancer tissues and cells. Overexpression of mTOR promoted cell growth, migration, and invasion, whereas mTOR knockdown diminished these phenomena in CRC cells in vitro. In addition, rhein directly targeted mTOR and inhibited the mTOR signaling pathway in CRC cells. Rhein promoted mTOR degradation through the ubiquitin-proteasome pathway. Intraperitoneal administration of rhein inhibited HCT116 xenograft tumor growth through the mTOR pathway. In conclusion, rhein exerts anticancer activity in vitro and in vivo by targeting mTOR and inhibiting the mTOR signaling pathway in CRC. Our results indicate that rhein is a potent anticancer agent that may be useful for the prevention and treatment of CRC.

A novel class of inhibitors that target SRSF10 and promote p53-mediated cytotoxicity on human colorectal cancer cells.

The elevated expression of the splicing regulator SRSF10 in metastatic colorectal cancer (CRC) stimulates the production of the pro-tumorigenic BCLAF1-L splice variant. We discovered a group of small molecules with an aminothiazole carboxamide core (GPS167, GPS192 and others) that decrease production of BCLAF1-L. While additional alternative splicing events regulated by SRSF10 are affected by GPS167/192 in HCT116 cells (e.g. in MDM4, WTAP, SLK1 and CLK1), other events are shifted in a SRSF10-independent manner (e.g. in MDM2, NAB2 and TRA2A). GPS167/192 increased the interaction of SRSF10 with the CLK1 and CLK4 kinases, leading us to show that GPS167/192 can inhibit CLK kinases preferentially impacting the activity of SRSF10. Notably, GPS167 impairs the growth of CRC cell lines and organoids, inhibits anchorage-independent colony formation, cell migration, and promotes cytoxicity in a manner that requires SRSF10 and p53. In contrast, GPS167 only minimally affects normal colonocytes and normal colorectal organoids. Thus, GPS167 reprograms the tumorigenic activity of SRSF10 in CRC cells to elicit p53-dependent apoptosis.

Evaluating the Epithelial-Mesenchymal Program in Human Breast Epithelial Cells Cultured in Soft Agar Using a Novel Macromolecule Extraction Protocol.

Simple SummaryAnchorage-independent soft agar colony formation assays have been widely used as an in vitro surrogate for in vivo tumour formation in xenograft studies, and has found much utility in studies such as cancer drug development. However, molecular characterisation of cells grown in soft agar has proven difficult and sometimes even impossible. We developed a set of new methods that allow DNA, RNA and proteins (including phosphoproteins) to be extracted from cells grown in soft agar, even without visible colony formation. We used these methods to demonstrate the role of the epithelial-mesenchymal program in the malignant transformation of a classical human mammary epithelial cell model.The ability to grow in anchorage-independent conditions is an important feature of malignant cells, and it is well-established that cellular phenotypes in adherent cultures can differ widely from phenotypes observed in xenografts and anchorage-independent conditions. The anchorage-independent soft-agar colony formation assay has been widely used as a bridge between adherent cell cultures and animal tumor studies, providing a reliable in vitro tool to predict the tumorigenicity of cancer cells. However, this functional assay is limited in its utility for molecular mechanistic studies, as currently there is no reliable method that allows the extraction of biological macromolecules from cells embedded in soft-agar matrices, especially in experimental conditions where no visible colonies form. We developed a set of new methods that enable the extraction of DNA, RNA and proteins directly from cells embedded in soft agar, allowing for a wide range of molecular signaling analysis. Using the new methods and human mammary epithelial cells (HMECs), we studied the role of epithelial-mesenchymal transition (EMT) in the ability of HMECs to form colonies in soft agar. We found that, when cultured in soft agar instead of in adherent cultures, immortalized non-malignant HME-hTERT cells upregulated the epithelial program, which was noted to be necessary for their survival in this anchorage-independent condition. Overexpression of SV40 small T antigen (ST) or the EMT master-regulator SNAI1 negates this requirement and significantly enhances colony formation in soft agar driven by mutant-RAS. Interestingly, we found that, similar to SNAI1, ST also promotes EMT changes in HMECs, providing further support for EMT as a prerequisite for the efficient anchorage-independent colony formation driven by mutant-RAS in our HMEC model.

S100A14 promotes progression and gemcitabine resistance in pancreatic cancer

S100 calcium binding protein A14 (S100A14) plays an important role in the progression of several types of cancer. However, its roles in pancreatic ductal adenocarcinoma (PDAC) are largely unexplored. Here, we characterized the functional roles of S100A14 in the progression and chemoresistance of PDAC. Gene expression microarray identified that S100A14 was significantly highly expressed in four pairs of human PDAC tumor compared with corresponding non-tumor tissues genes. Quantitative reverse transcription PCR (qRT-PCR), western blotting and immunohistochemical staining (IHC) showed that S100A14 was frequently overexpressed in PDAC cell lines and tissues. Moreover, expression level of S100A14 was positively correlated to advanced cancer stages. Further, Kaplan-Meier survival analysis suggested that PDAC patients with low S100A14 expression had longer overall survival in TCGA PDAC datasets. Transient overexpressing of S100A14 promoted cell proliferation, anchorage-independent colony formation, cell migration and invasion in cell lines with low endogenous S100A14 levels, while transient silencing of S100A14 inhibited cell proliferation, anchorage-independent colony formation, cell migration and invasion in cell lines with high endogenous S100A14 levels. Persistent knockdown of S100A14 by transducing shRNAs carrying lentivirus inhibited subcutaneous tumor formation in nude mice, and sensitized the PDAC cells to gemcitabine treatment. Taken together, S100A14 exhibited oncogenic properties by promoting cell proliferation, transformation, migration and invasion, and enhanced in vivo tumor growth. More importantly, inhibition of S100A14 could effectively abrogate the cancerous properties of the PDAC cells. Our study indicated that S100A14 was a valuable target for the development of therapeutic strategy, as well as a diagnostic and prognosis biomarker for PDAC patients.

PEPCK-M recoups tumor cell anabolic potential in a PKC-\u03b6-dependent manner

BackgroundMitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M; PCK2) is expressed in all cancer types examined and in neuroprogenitor cells. The gene is upregulated by amino acid limitation and ER-stress in an ATF4-dependent manner, and its activity modulates the PEP/Ca2+ signaling axis, providing clear arguments for a functional relationship with metabolic adaptations for cell survival. Despite its potential relevance to cancer metabolism, the mechanisms responsible for its pro-survival activity have not been completely elucidated.Methods[U-13C]glutamine and [U-13C]glucose labeling of glycolytic and TCA cycle intermediates and their anabolic end-products was evaluated quantitatively using LC/MS and GC/MS in conditions of abundant glucose and glucose limitation in loss-of-function (shRNA) and gain-of-function (lentiviral constitutive overexpression) HeLa cervix carcinoma cell models. Cell viability was assessed in conjunction with various glucose concentrations and in xenografts in vivo.ResultsPEPCK-M levels linearly correlated with [U-13C]glutamine label abundance in most glycolytic and TCA cycle intermediate pools under nutritional stress. In particular, serine, glycine, and proline metabolism, and the anabolic potential of the cell, were sensitive to PEPCK-M activity. Therefore, cell viability defects could be rescued by supplementing with an excess of those amino acids. PEPCK-M silenced or inhibited cells in the presence of abundant glucose showed limited growth secondary to TCA cycle blockade and increased ROS.In limiting glucose conditions, downregulation of PKC-ζ tumor suppressor has been shown to enhance survival. Consistently, HeLa cells also sustained a survival advantage when PKC-ζ tumor suppressor was downregulated using shRNA, but this advantage was abolished in the absence of PEPCK-M, as its inhibition restores cell growth to control levels. The relationship between these two pathways is also highlighted by the anti-correlation observed between PEPCK-M and PKC-ζ protein levels in all clones tested, suggesting co-regulation in the absence of glucose. Finally, PEPCK-M loss negatively impacted on anchorage-independent colony formation and xenograft growth in vivo.ConclusionsAll in all, our data suggest that PEPCK-M might participate in the mechanisms to regulate proteostasis in the anabolic and stalling phases of tumor growth. We provide molecular clues into the clinical relevance of PEPCK-M as a mechanism of evasion of cancer cells in conditions of nutrient stress.