Inhibition Of Anchorage-independent Growth Research Articles

Sustained use of antimutagenic bioactives having affinity with DNA minor groove could prevent in vitro neoplastic induction

IntroductionStudies on the antimutagenic and anti-neoplastic effect of dietary ingredients can be useful in developing nutraceuticals for preventing neoplastic induction. In the current study the query whether food rich in antimutagenic potential equally suppress the neoplastic induction, was addressed, with potential mechanism of action. MethodsThe antimutagenic activity was evaluated using the TK6 gene mutation assay, assessing the reduction in ethyl methanesulfonate (EMS)-induced mutation in presence of vegetable extracts. The anti-⁠neoplastic activity was assessed using the C3H/⁠10T1/⁠2 cell transformation assay, quantifying the inhibition of anchorage-independent growth and type III foci formation. The prominent compounds in the vegetable extract were characterised using gas chromatography-⁠mass spectrometry (GC-⁠MS), and their interactions with DNA were predicted using molecular docking. ResultsAmong the vegetable extracts, garlic exhibited the most potent antimutagenic and anti-neoplastic effects, followed by spinach and beans. Garlic extract exhibited significantly high antimutagenic potential (59±3%), and anti-neoplastic activity in the terms of inhibition of anchorage independence (65±3%) and inhibition of type III foci formation (90±5%). GC-MS analysis of garlic extract identified abundant organosulphur compounds, terpenes, and terpenoids. Molecular docking studies suggested that these compounds bind to the DNA minor groove with binding energies ranging from −⁠4.3 to −⁠7.21 kcal mol-⁠1. DiscussionThe antimutagenic and antineoplastic activities showed a positive correlation (r=0.92–0.93), where garlic extract showed the highest potential. The DNA binding properties of the prominent compounds in garlic extract suggests a potential mechanism of action involving interference with carcinogen activity and subsequent prevention of gene dysregulation.

Isorhapontigenin inhibition of basal muscle-invasive bladder cancer attributed to its downregulation of SNHG1 and DNMT3b

BackgroundBladder cancer (BC) is among the most prevalent malignant urothelial tumors globally, yet the prognosis for patients with muscle-invasive bladder cancer (MIBC) remains dismal, with a very poor 5-year survival rate. Consequently, identifying more effective and less toxic chemotherapeutic alternatives is critical for enhancing clinical outcomes for BC patients. Isorhapontigenin (ISO), a novel stilbene isolated from a Gnetum found in certain provinces of China, has shown potential as an anticancer agent due to its diverse anticancer activities. Despite its promising profile, the specific anticancer effects of ISO on BC and the underlying mechanisms are still largely unexplored.MethodsThe anchorage-independent growth, migration and invasion of BC cells were assessed by soft agar and transwell invasion assays, respectively. The RNA levels of SOX2, miR-129 and SNHG1 were quantified by qRT-PCR, while the protein expression levels were validated through Western blotting. Furthermore, methylation-specific PCR was employed to assess the methylation status of the miR-129 promoter. Functional assays utilized siRNA knockdown, plasmid-mediated overexpression, and chemical inhibition approaches.ResultsOur study demonstrated that ISO treatment significantly reduced SNHG1 expression in a dose- and time-dependent manner in BC cells, leading to the inhibition of anchorage-independent growth and invasion in human basal MIBC cells. This effect was accompanied by the downregulation of MMP-2 and MMP-9 and the upregulation of the tumor suppressor PTEN. Further mechanistic investigations revealed that SOX2, a key upstream regulator of SNHG1, played a crucial role in mediating the ISO-induced transcriptional suppression of SNHG1. Additionally, we found that ISO treatment led to a decrease in DNMT3b protein levels, which in turn mediated the hypomethylation of the miR-129 promoter and the subsequent suppression of SOX2 mRNA 3’-UTR activity, highlighting a novel pathway through which ISO exerts its anticancer effects.ConclusionsCollectively, our study highlights the critical role of SNHG1 downregulation as well as its upstream DNMT3b/miR-129/SOX2 axis in mediating ISO anticancer activity. These findings not only elucidate the mechanism of action of ISO but also suggest novel targets for BC therapy.

Abstract PO3-25-10: Selective and Effective Targeting of Triple-Negative Breast Cancer by CMPD1: Inhibition of MAP kinase-activated protein kinase 2 and Microtubule Dynamics

Abstract Background: Microtubule-targeting agents (MTAs) have been widely utilized in cancer treatment, but the development of novel MTAs has faced limitations. CMPD1, a highly selective inhibitor of MAP kinase-activated protein kinase 2 (MK2) with dual functions as a microtubule inhibitor, shows promise as a targeted therapy for triple-negative breast cancer (TNBC). This study aimed to investigate the effects of CMPD1, targeting MK2 and acting as a microtubule inhibitor, on cell cycle progression, microtubule dynamics, and TNBC-related processes. Methods: CMPD1 was evaluated in non-transformed retinal pigment epithelial cells (RPE1) and TNBC cell lines, including MDA-MB-231 and MDA-MB-468 cells. Time-lapse microscopy and immunofluorescence staining were employed to observe the effects of CMPD1 on mitotic progression, cellular morphology, activation of the spindle assembly checkpoint (SAC), and microtubule dynamics. Anchorage-independent growth assays and in vivo studies using a TNBC xenograft model were performed to assess the impact of CMPD1 on colony formation, tumor growth, and metastasis. Results: CMPD1 treatment induced prometaphase arrest and mitotic slippage in both non-transformed and TNBC cells. This arrest was associated with SAC activation, evidenced by increased levels of Mad1 at kinetochores. CMPD1 exhibited robust microtubule depolymerization, primarily targeting microtubule plus-ends and inhibiting microtubule polymerization in vitro. Furthermore, CMPD1 disrupted the organization of the microtubule network in interphase cells and perturbed the mitotic spindle during mitosis. Additionally, CMPD1 treatment resulted in the formation of cytoplasmic protrusions, membrane deformations, and dynamic movement of clustered mitotic chromosomes. These morphological alterations were indicative of the impact of CMPD1 on cellular structures and processes during mitosis. Notably, CMPD1 demonstrated significant efficacy in inhibiting anchorage-independent growth in TNBC cells, highlighting its potential as an anti-TNBC agent. In the TNBC xenograft model, CMPD1 exhibited superior anti-tumor activity compared to the standard chemotherapeutic agent Taxol. Tumor growth was markedly suppressed, and the formation of metastatic lesions was significantly reduced upon CMPD1 treatment. Furthermore, CMPD1 treatment effectively inhibited TNBC cell migration and invasion, likely through its modulation of microtubule dynamics. The disruption of microtubule dynamics by CMPD1 impaired the ability of TNBC cells to migrate and invade surrounding tissues, which are crucial processes in cancer metastasis. Importantly, CMPD1 showed high selectivity for TNBC cells, exerting minimal cytotoxic effects on normal cells, and could be efficiently washed out. This selectivity and washout ability enhance its potential as a targeted therapeutic agent for TNBC treatment. Conclusions: CMPD1, a highly selective MK2 inhibitor and microtubule inhibitor, exerts profound effects on cell cycle progression, microtubule dynamics, and TNBC-related processes. It induces prometaphase arrest, mitotic slippage, and microtubule depolymerization, resulting in altered cellular morphology. CMPD1 displays high selectivity for TNBC cells, while sparing normal cells and allowing efficient washout, making it a promising therapeutic agent for TNBC treatment. Its inhibition of anchorage-independent growth, suppression of tumor growth in vivo, and ability to impair TNBC cell migration and invasion further underscore its potential as a targeted anti-TNBC therapy. Citation Format: Mamoru Takada, Yu-Chia Chen, Yu Muhan, Hideyuki Yamada, Junta Sakakibara, Hiroshi Fujimoto, Takeshi Nagashima, Masayuki Otsuka, Motoki Takaku, Aussie Suzuki. Selective and Effective Targeting of Triple-Negative Breast Cancer by CMPD1: Inhibition of MAP kinase-activated protein kinase 2 and Microtubule Dynamics [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-25-10.

PRELP Regulates Cell-Cell Adhesion and EMT and Inhibits Retinoblastoma Progression.

Simple SummaryMutation of the RB1 tumor suppressor gene is fundamental in retinoblastoma initiation and progression although its downstream mechanism has not been well elucidated. Here, we found that expression of proline/arginine-rich end leucine-rich repeat protein (PRELP) is strongly downregulated in human retinoblastoma and highly expressed in Müller glial cells in normal retina. Deletion of PRELP in mice resulted in retinal dysplasia associated with enhanced proliferation. mRNA expression profiling revealed that cancer pathways were strongly activated in PRELP−/− retina. Additionally, cell–cell adhesion was inhibited while epithelial mesenchymal transition (EMT) and inflammation were activated. On the other hand, application of PRELP protein to retinoblastoma cell lines enhances cell–cell and cell–substrate adhesion and inhibits anchorage independent growth by reversing EMT. These observations indicate that PRELP downregulation in human retinoblastoma can contribute cancer progression through regulation of cell adhesion and EMT suggested that PRELP application might be a novel strategy for retinoblastoma treatment.Retinoblastoma (RB) is the most common intraocular pediatric cancer. Nearly all cases of RB are associated with mutations compromising the function of the RB1 tumor suppressor gene. We previously demonstrated that PRELP is widely downregulated in various cancers and our in vivo and in vitro analysis revealed PRELP as a novel tumor suppressor and regulator of EMT. In addition, PRELP is located at chromosome 1q31.1, around a region hypothesized to be associated with the initiation of malignancy in RB. Therefore, in this study, we investigated the role of PRELP in RB through in vitro analysis and next-generation sequencing. Immunostaining revealed that PRELP is expressed in Müller glial cells in the retina. mRNA expression profiling of PRELP−/− mouse retina and PRELP-treated RB cells found that PRELP contributes to RB progression via regulation of the cancer microenvironment, in which loss of PRELP reduces cell–cell adhesion and facilitates EMT. Our observations suggest that PRELP may have potential as a new strategy for RB treatment.

Abstract P082: Distinct and significant anti-cancer efficacy of plinabulin in patient derived small cell lung cancer (SCLC) 3D soft agar clonogenic assays

Abstract Plinabulin (Plin) is a new chemical entity and a selective immunomodulating microtubule-targeting agent (SIMBA), which exerts immune-enhancing effects through increasing dendritic cell (DC) maturation and DC-dependent antigen presentation to CD4+ and CD8+ T-Cells. Here we evaluated whether Plin also exerts direct anti-cancer effects against approximately 80 patient derived (PDX) models. Plin was characterized for its ability to inhibit anchorage independent growth and ex vivo colony formation of PDX tumor cells in semi-solid medium. The compound was investigated in PDX models of various cancer types, using a 3D clonogenic assay in a 96-well format. Briefly, tumor cell suspensions were prepared from subcutaneous xenografts in NMRI nu/nu mice and seeded into 96 well ultra-low attachment plates within a layer of semi-solid medium. Plin was added to the assay plate at either day 2 or day 7 and tested at 9 concentrations in half-log increments up to a concentration of 3 µM. Supernatant was exchanged 24 h after dosing and cells were further incubated at 37°C and 7.5 % CO2 up to a total assay time of 8 or 13 days. Colony counts based on image analysis were utilized to evaluate efficacy. Plin inhibited tumor colony formation in a concentration-dependent manner. With Plin treatment beginning 2 days after PDX cell seeding, the most sensitive cancer types were small cell lung cancer (mean absolute IC70 = 0.035 μM; n = 7), bladder cancer (mean absolute IC70 = 0.038 μM; n = 9), and soft tissue sarcoma (mean absolute IC70 = 0.057 μM; n = 10). Melanoma models were observed to be the least sensitive to Plin monotherapy, with a mean absolute IC70 of 1.105 μM (n = 19). With Plin treatment beginning 7 days after PDX cell seeding, based on absolute IC70 values, 9 out of 68 tumor models were sensitive towards plinabulin. The most sensitive histotypes were gastric cancer (Asian, mean abs. IC70 = 0.319 µM, n = 3), small cell lung cancer (mean abs. IC70 = 0.385 µM, n = 7; 3 below 50 nM), osteosarcoma (mean abs. IC70 = 0.624 µM, n = 3), and central nervous system cancer (mean abs. IC70 = 1.521 µM, n = 6). Overall, the most responsive models based on IC70 were the small cell lung cancer models 2156 (abs. IC70 = 0.015 µM), 1129 (abs. IC70 = 0.032 µM) and 650 (abs. IC70 = 0.032 µM), and the osteosarcoma model 1186 (abs. IC70 = 0.027 µM). Soft tissue sarcoma models (n = 8), Her2-enriched breast cancer models (n = 6), melanoma models (n = 9), bladder cancer models (n = 6), and gastric cancer models (Caucasian, n = 3) were observed to be the least sensitive cancer types with a mean IC70 value > 3 µM. In conclusion, using patient-derived tumor models in a 3D clonogenic assay, SCLC tumor types were the most sensitive to Plin. These results provide mechanistic support for the preliminary positive results recently reported with Plin in combination with Nivolumab and Ipilimumab in SCLC patients (Malhotra ASCO 2021). Citation Format: George K. Lloyd, James Tonra, Claudia Goettlich, Tobias Deigner, Ramon Mohanlal, Lan Huang. Distinct and significant anti-cancer efficacy of plinabulin in patient derived small cell lung cancer (SCLC) 3D soft agar clonogenic assays [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P082.

Identification of Thieno[3,2-d]pyrimidine Derivatives as Dual Inhibitors of Focal Adhesion Kinase and FMS-like Tyrosine Kinase 3.

Focal adhesion kinase (FAK) is overexpressed in highly invasive and metastatic cancers. To identify novel FAK inhibitors, we designed and synthesized various thieno[3,2-d]pyrimidine derivatives. An intensive structure-activity relationship (SAR) study led to the identification of 26 as a lead. Moreover, 26, a multitargeted kinase inhibitor, possesses excellent potencies against FLT3 mutants as well as FAK. Gratifyingly, 26 remarkably inhibits recalcitrant FLT3 mutants, including F691L, that cause drug resistance. Importantly, 26 is superior to PF-562271 in terms of apoptosis induction, anchorage-independent growth inhibition, and tumor burden reduction in the MDA-MB-231 xenograft mouse model. Also, 26 causes regression of tumor growth in the MV4-11 xenograft mouse model, indicating that it could be effective against acute myeloid leukemia (AML). Finally, in an orthotopic mouse model using MDA-MB-231, 26 remarkably prevents metastasis of orthotopic tumors to lymph nodes. Taken together, the results indicate that 26 possesses potential therapeutic value against highly invasive cancers and relapsed AML.

Pleiotropic effects of probenecid on three-dimensional cultures of prostate cancer cells

AimsChemoresistance remains a persistent challenge in advanced prostate cancer therapy. Probenecid reportedly inhibits multiple drug-efflux transporters; hence, it can be employed as a potential sensitizer for chemotherapy. In the present study, we evaluated the effects of probenecid on three-dimensional (3D)-cultures of prostate cancer cells. Main methodsProstate cancer cell lines, 22Rv1 and PC-3 were cultured as multicellular tumor spheroids. The effects of probenecid were evaluated using the MTT assay for viability, microscopy for spheroid size, and soft agar colony formation assay for anchorage-independent growth. Key findingsThe 3D-cultured 22Rv1 cells were less sensitive to cisplatin and doxorubicin than two-dimensional (2D) cell culture. Co-administration of probenecid at a low (100 or 300 μM), but not high (500 μM), concentration increased the sensitivity to cisplatin or doxorubicin in 22Rv1 spheroids. Probenecid increased the expression of ABCG2, a multidrug resistance transporter, in a dose-dependent manner. Furthermore, treatment with probenecid alone reduced the growth of 22Rv1 spheroids. Conversely, probenecid inhibited spheroid compaction rather than growth inhibition in 3D-cultured PC-3 cells. Moreover, probenecid inhibited colony formation of 22Rv1 and PC-3 cells in soft agar, as well as downregulated focal adhesion kinase (FAK), a crucial factor in anchorage-independent growth. SignificanceIn 3D-cultured prostate cancer cells, probenecid demonstrated pleiotropic effects such as chemosensitization, growth suppression, inhibition of spheroid compaction, and suppression of anchorage-independent growth. Elucidating the detailed mechanism underlying these probenecid actions could result in the identification of novel therapeutic targets toward the advanced prostate cancer.

Vaccinium myrtillus L. extract and its native polyphenol-recombined mixture have anti-proliferative and pro-apoptotic effects on human prostate cancer cell lines.

Vaccinium myrtillus berry extract (VME) and a recombined standard mixture (RSM) of its main native phenolic compounds were investigated for cell growth inhibition and pro-apoptotic activity on hormone-dependent (LNCaP) and hormone-independent (PC3 and DU-145) prostate cancer (PCa) cell lines. Normal prostate epithelial cells (PrEC) were also studied in comparison. VME hindered anchorage-dependent PCa cell proliferation in a dose-dependent manner, that is, at 1/800 (v/v) dilution for LNCaP and PC3, and 1/100 (v/v) dilution for DU-145 (corresponding to 14.15 and 113.2 μg cyanidin-3-O-glucoside equivalents per ml of culture medium), respectively. VME had a growth inhibitory effect towards PrEC at the same dilution of DU-145 cells although the IC50 values indicated that PrEC are more resistant than PCa cell lines. VME also reduced the anchorage-independent growth of PCa cells. The study of the apoptotic profile (i.e., non-apoptotic, early apoptotic, late apoptotic and necrotic cells) evidenced that the apoptotic rate (early+late) was statistically higher in all three cell lines exposed to VME compared to control. Anchorage-dependent and anchorage-independent growth inhibition of RSM was very similar to that displayed by VME. Moreover, RSM exerted its growth inhibitory effect also under hypoxia, the latter representing a biological condition known to sustain PCa proliferation and aggressiveness.

Abstract 4806: SIRT5 inhibition causes increased oxidative stress and impairs tumor progression and metastasis

Abstract Cancer cells undergo metabolic reprogramming, producing energy through aerobic glycolysis followed by lactic acid fermentation. Consequently, targeting key metabolic enzymes has emerged as a potential avenue to selectively inhibit tumor growth. The sirtuin family of deacylases has roles in both metabolism and cancer. The mitochondrial sirtuin SIRT5 regulates protein post-translational modifications on metabolic enzymes by catalyzing the removal of succinyl, malonyl, and glutaryl moieties. SIRT5 is over-expressed in many cancers, including breast cancer. Therefore, we hypothesized that SIRT5 promotes breast cancer progression by facilitating metabolic reprogramming. SIRT5 knockdown in human cancer cells inhibited anchorage independent growth but had little effect on non-transformed cells. To examine how SIRT5 loss impacts tumorigenesis in vivo, we utilized MMTV-PyMT transgenic mice, which are prone to mammary adenocarcinoma and lung metastasis. SIRT5 knockout (KO) MMTV-PyMT mice had increased survival, decreased tumor size, and reduced lung metastases, as compared to SIRT5 wild-type (WT) MMTV-PyMT controls. SIRT5 KO cancer cells had higher levels of reactive oxygen species and displayed lower levels of important antioxidants such as NADPH and GSH. These results suggest that SIRT5 KO cells are more susceptible to oxidative stress, hinting that SIRT5 may be promoting breast cancer by mitigating ROS through one or more of its key metabolic substrates. To determine the therapeutic potential of SIRT5 inhibition, we created potent and specific small molecule SIRT5 inhibitors. Pharmacological inhibition of SIRT5 impaired mammary tumor growth in both transgenic and human breast cancer xenograft mouse models, with no apparent toxicity. Considering that Sirt5 knockout mice are generally normal, with only mild phenotypes observed, these data establish SIRT5 as a promising target for treating breast cancer. Citation Format: Irma Fernandez, Yashira N. Abril, Jun Y. Hong, Min Yang, Sushabhan Sadhukhan, Hening Lin, Robert Weiss. SIRT5 inhibition causes increased oxidative stress and impairs tumor progression and metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4806.

Preclinical Efficacy and Involvement of AKT, mTOR, and ERK Kinases in the Mechanism of Sulforaphane against Endometrial Cancer.

Sulforaphane exerts anti-cancer activity against multiple cancer types. Our objective was to evaluate utility of sulforaphane for endometrial cancer therapy. Sulforaphane reduced viability of endometrial cancer cell lines in association with the G2/M cell cycle arrest and cell division cycle protein 2 (Cdc2) phosphorylation, and intrinsic apoptosis. Inhibition of anchorage-independent growth, invasion, and migration of the cell lines was associated with sulforaphane-induced alterations in epithelial-to-mesenchymal transition (EMT) markers of increased E-cadherin and decreased N-cadherin and vimentin expression. Proteomic analysis identified alterations in AKT, mTOR, and ERK kinases in the networks of sulforaphane effects in the Ishikawa endometrial cancer cell line. Western blots confirmed sulforaphane inhibition of AKT, mTOR, and induction of ERK with alterations in downstream signaling. AKT and mTOR inhibitors reduced endometrial cancer cell line viability and prevented further reduction by sulforaphane. Accumulation of nuclear phosphorylated ERK was associated with reduced sensitivity to the ERK inhibitor and its interference with sulforaphane activity. Sulforaphane induced apoptosis-associated growth inhibition of Ishikawa xenograft tumors to a greater extent than paclitaxel, with no evidence of toxicity. These results verify sulforaphane’s potential as a non-toxic treatment candidate for endometrial cancer and identify AKT, mTOR, and ERK kinases in the mechanism of action with interference in the mechanism by nuclear phosphorylated ERK.

Abstract 2013: A carbonic anhydrase inhibitor methazolamide potentiates efficacy of gemcitabine by modulating anti-proliferative activity and cancer stem cell markers in pancreatic carcinoma

Abstract Pancreatic cancer is the fourth cause for cancer related deaths globally. By 2020, incidence of new cases is expected to be 420,000 with equal number of associated mortalities. Overall 5-year survival has marginally improved over past despite of our growing understanding of the disease. Surgery remains standard of care for resectable tumors followed by adjuvant chemotherapy which resulted in median survival of 26 months. Newer therapies targeting stroma and immunotherapies hold promise for the future. Noticeably, a high unmet medical need warrant novel therapy to extend survival over a year in metastatic settings. Drug repositioning holds promise over the conventional drug discovery, as it reduces development time and cost. Using this approach, we report promising anticancer activity of Methazolamide (MZM) in pancreatic cancer. MZM, a synthetic derivative of sulphonamide and a carbonic anhydrase IX (CA) inhibitor is used for the treatment of ocular conditions for lowering intraocular pressure. Overexpression of CA is implicated in acidification of tumor microenvironment leading to hypoxia which in turn promotes growth and metastasis of the tumor. Therefore, modulation of extracellular tumor pH via inhibition of CA represents a promising course to anticancer therapy. Here, we report anti-proliferative activity of MZM in 12 pancreatic cell lines including patient derived (PDX) in 2D models and anchorage independent growth inhibition in 3D models. Combination treatment with GEM yielded significant growth inhibition than drug alone in all tested cell lines. Additive effects were observed for the combination in PDX-1986, PDX-546, Capan-2, Mia-Pa-Ca and PANC-1 cells. Further, in vivo efficacy of MZM in combination with GEM was performed using PDX-546 in nude mice. Combination group showed significant tumor growth inhibition (72%, p<0.05) as against drug alone, MZM at 60 mg/kg, p.o. (50%, p<0.05) and GEM at 50 mg/kg i.v. (61%, p<0.05). There was no significant body weight loss. For PK/PD studies, MZM levels were measured in tumors and PD effect was evaluated using various markers in tumor samples. Expression of target genes was measured by qRT-PCR. Significant reduction in expression of stem cell markers viz. RAC1, OCT4, CD34, CD14, Nanog and proliferation marker (Ki67) was observed in combination group as compared to drug alone. In addition, anti-angiogenic potential viz. would healing, tube formation and expression of important anti-angiogenic markers like HIF1α, VEGF were profoundly modulated in MZM and combination group. Thus, our study indicates that the combination of MZM, a CA inhibitor with GEM is a promising novel approach for the treatment of pancreatic cancer especially in metastatic settings. Citation Format: Kalpana Joshi, Jeevan D. Ghosalkar, Vinay R. Sonawane, Siddhika R. Raut, Geena Malhotra. A carbonic anhydrase inhibitor methazolamide potentiates efficacy of gemcitabine by modulating anti-proliferative activity and cancer stem cell markers in pancreatic carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2013.

New compound ChlA-F induces autophagy-dependent anti-cancer effect via upregulating Sestrin-2 in human bladder cancer

ChlA-F is a novel conformation-derivative of Cheliensisin A, styryl-lactone isolates that show potent anti-tumor potential in vivo and vitro. However, the anti-cancer activity and its potential mechanisms underlying ChlA-F action have never been explored. In the present study, we evaluated the potency of ChlA-F on autophagy-mediated anchorage-independent growth inhibition in human high-grade invasive bladder cancer (BC) cells. We found that ChlA-F treatment significantly inhibited anchorage-independent growth of human BC cells by inducing autophagy in a Sestrin-2 (SESN2)-dependent fashion. Our results revealed that ChlA-F treatment specifically induced SESN2 expression via increasing its transcription and mRNA stability. On one hand, ChlA-F treatment markedly attenuated Dicer protein abundance, in turn abolishing miR-27a maturation and further relieving miR-27a binding directly to SESN2 mRNA 3′UTR, thereby promoting SESN2 mRNA stabilization. On the other hand, ChlA-F treatment promoted Sp1 abundance and consequently mediated SESN2 transcription. These results demonstrate that its activation of the autophagic pathway through specifically promoting SESN2 expression mediates the anti-cancer effect of ChlA-F, which offers insights into the novel anti-cancer effect of ChlA-F on BC, as well as providing therapeutic alternatives against human BC.

GRWD1 regulates ribosomal protein L23 levels via the ubiquitin-proteasome system.

Glutamate-rich WD40 repeat-containing 1 (GRWD1) is a Cdt1-binding protein that promotes mini-chromosome maintenance (MCM) loading through its histone chaperone activity. GRWD1 acts as a tumor-promoting factor by downregulating p53 (also known as TP53) via the RPL11-MDM2-p53 axis. Here, we identified GRWD1-interacting proteins using a proteomics approach and showed that GRWD1 interacts with various proteins involved in transcription, translation, DNA replication and repair, chromatin organization, and ubiquitin-mediated proteolysis. We focused on the ribosomal protein ribosomal protein L23 (RPL23), which positively regulates nucleolar stress responses through MDM2 binding and inhibition, thereby functioning as a tumor suppressor. Overexpression of GRWD1 decreased RPL23 protein levels and stability; this effect was restored upon treatment with the proteasome inhibitor MG132. EDD (also known as UBR5), an E3 ubiquitin ligase that interacts with GRWD1, also downregulated RPL23, and the decrease was further enhanced by co-expression of GRWD1. Conversely, siRNA-mediated GRWD1 knockdown upregulated RPL23. Co-expression of GRWD1 and EDD promoted RPL23 ubiquitylation. These data suggest that GRWD1 acts together with EDD to negatively regulate RPL23 via the ubiquitin-proteasome system. GRWD1 expression reversed the RPL23-mediated inhibition of anchorage-independent growth in cancer cells. Our data suggest that GRWD1-induced RPL23 proteolysis plays a role in downregulation of p53 and tumorigenesis.

Abstract 2306: Transcriptional and post-transcriptional upregulation of p27 mediates growth inhibition of isorhapontigenin/ISO on human bladder cancer cells

Abstract Our recent studies have demonstrated that Isorhapontigenin (ISO), a new derivative isolated from the Chinese herb Gnetum Cleistostachyum, effectively induces cell-cycle arrest at the G0/G1 phase and inhibits anchorage-independent cell growth through the miR-137/Sp1/cyclin D1 axis in human muscle invasive bladder cancer cells both in vitro and in vivo. Herein, we found that treatment of muscle invasive bladder cancer cells with ISO resulted in a significant upregulation of p27, a key cyclin-dependent kinase inhibitor. Importantly, knockdown of p27 caused a decline in the ISO-induced G0-G1 growth arrest and reversed ISO suppression of anchorage-independent growth in bladder cancer cells. Mechanistic studies revealed that ISO promoted p27 expression at mRNA transcription level through increasing direct binding of FOXO1 to its promoter, while knockdown of FOXO1 attenuated ISO inhibition of cell growth. On the other hand, ISO upregulated the 3'UTR activity of p27, which was accompanied by a reduction of miR-182 expression. In line with these observations, ectopic expression of miR-182 significantly blocked p27 3'UTR activity, whereas mutation of the miR-182 binding site at p27 mRNA 3'UTR effectively reversed this inhibition. Accordingly, ectopic expression of miR-182 also attenuated ISO upregulation of p27 expression and impaired ISO inhibition of cancer cell growth. Our results not only provide novel insight into understanding of the underlying mechanism related to regulation of muscle invasive bladder cancer cell growth, but also identify new roles and mechanisms underlying ISO inhibition of the growth of human bladder cancer cells. Citation Format: Jiheng Xu, Jingxia Li, Chuanshu Huang, Haishan Huang, Guosong Jiang. Transcriptional and post-transcriptional upregulation of p27 mediates growth inhibition of isorhapontigenin/ISO on human bladder cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2306.

Regulation of AMPK-related glycolipid metabolism imbalances redox homeostasis and inhibits anchorage independent growth in human breast cancer cells

Breast cancer is one of the most lethal tumors in the world, among which 15% are triple-negative breast cancers (TNBCs) with higher metastasis and lower survival rate. Anoikis resistance is a key process during tumor metastasis, which is usually accompanied with metabolism reprogram. In this study, we established an anchorage independent growth model for MDA-MB-231 cells and investigated the changes in metabolism and redox homeostasis. Results showed that during detached-growth, MDA-MB-231 cells tend to generate ATP through fatty acid oxidation (FAO), instead of glycolysis. Amount of glucose was used for pentose phosphate pathway (PPP) to keep redox balance. Moreover, we discovered that a synthesized flavonoid derivative GL-V9, exhibited a potent inhibitory effect on the anchorage independent growth of TNBCs in vitro and anti-metastasis effect in vivo. In terms of the mechanism, GL-V9 could promote the expression and activity of AMPK, leading to the decrease of G6PD and the increase of p-ACC. Thus, the level of PPP was suppressed, whereas FAO was highly enhanced. The reprogram of glycolipid metabolism destroyed the redox balance ultimately and induced cell death. This paper indicated a novel regulating mechanism of redox homeostasis involving with glycolipid metabolism, and provided a potential candidate for the anti-metastatic therapy of TNBCs.

Lp16-PSP, a Member of YjgF/YER057c/UK114 Protein Family Induces Apoptosis and p21WAF1/CIP1 Mediated G1 Cell Cycle Arrest in Human Acute Promyelocytic Leukemia (APL) HL-60 Cells

Lp16-PSP (Latcripin 16-Perchloric acid Soluble Protein) from Lentinula edodes strain C91-3 has been reported previously in our laboratory to have selective cytotoxic activity against a panel of human cell lines. Herein, we have used several parameters in order to characterize the Lp16-PSP-induced cell death using human acute promyeloid leukemia (HL-60) as a model cancer. The results of phase contrast microscopy, nuclear examination, DNA fragmentation detection and flow cytometry revealed that high doses of Lp16-PSP resulted in the induction of apoptosis in HL-60 cells. The colorimetric assay showed the activation of caspase-8, -9, and -3 cascade highlighting the involvement of Fas/FasL-related pathway. Whereas, Western blot revealed the cleavage of caspase-3, increased expression of Bax, the release of cytochrome c and decreased expression of Bcl-2 in a dose-dependent manner, suggesting the intrinsic pathway might be involved in Lp16-PSP-induced apoptosis as well. Low doses of Lp16-PSP resulted in the anchorage-independent growth inhibition, induction of G1 phase arrest, accompanied by the increased expression of p21WAF1/CIP1, along with the decreased expression of cyclin D, E, and cdk6. In addition, Lp16-PSP resulted in constitutive translocation inhibition of transcription factor nuclear factor kappa B (NF-κB) into the nucleus by decreasing the phosphorylation of IκBα. All these findings suggested Lp16-PSP as a potential agent against acute promyeloid leukemia; however, further investigations are ultimately needed.

The tumour suppressor, miR-137, inhibits malignant melanoma migration by targetting the TBX3 transcription factor

The transcription factor, TBX3, is a key driver of malignant melanoma and any drug that impacts its expression is likely to have an impact on the treatment of this highly aggressive and treatment resistant cancer. Replacement of miRNAs that target oncogenes has gained much attention as a therapy because it is anticipated to be effective with little side-effects since miRNAs are naturally occurring and often target large set of genes in the same oncogenic pathway. Here we show that miR-137 levels correlate inversely with TBX3 mRNA levels in a panel of melanoma cell lines and in a cohort of patients with primary melanoma. Low levels of miR-137 and high levels of TBX3 are shown to be associated with poor patient survival. We show that miR-137 binds a conserved site in the TBX3 3′ untranslated region and that a miR-137 mimic significantly reduces endogenous levels of TBX3 and inhibits anchorage independent growth and migration of malignant melanoma cells. Novel data are provided that the miR-137/TBX3/E-cadherin axis plays an important role in melanomagenesis and that miR-137 replacement is a potential therapeutic approach for treating melanomas.

Abstract 119: Vitamin K2 targets castration-resistant prostate cancer VCaP cells by reactive oxygen species mediated apoptotic cell death

Abstract Prostate Cancer (PCa) is the second most common cancer in western countries especially in US population, in which castration-resistant prostate cancer (CRPC) is the major cause for patient mortality. Current treatment options available for CRPC are not efficient and have undesirable side effects. Hence there is an urgent need to develop non-toxic and effective treatment strategies for CRPC. Vitamin K2 (VK2), a natural menaquinone has several medicinal values including anti-cancer activity and anti-osteoporosis effect. The aim of this study is to evaluate the therapeutic effects of Vitamin K2 (VK2) and its anti-cancer mechanism against CRPC. In this study, we have used VCaP cell line (ATCC) which is established from a patient with hormone refractory prostate cancer. VCaP cells were treated with various concentrations of VK2 to evaluate its effects on cell viability by MTT assay, anchorage independent growth by soft agar assay, cellular senescence by beta-galactosidase staining assay and cancer cell migration by wound healing assay. We have also assessed the VK2-induced production of intracellular reactive oxygen species (ROS) using DCF (2′,7′-dichlorofluorescein) probe based fluorescence assay. VK2 induced apoptosis was detected by Annexin-V FITC and TUNEL assays. Western blot analysis was utilized to uncover the anti-proliferative and anti-metastatic mechanisms of VK2 against CRPC. Our results showed that VK2 significantly inhibits the proliferation of VCaP cells in a dose dependent manner at 48 hrs treatment in vitro. MTT data also showed that anti-proliferative effects of VK2 were significantly abrogated in the presence of anti-oxidant N-acetyl cysteine (NAC) and caspase inhibitor Z-VAD-FMK suggesting that ROS and caspase activation as the underlying anti-cancer mechanisms of VK2 in CRPC cells. In addition, VK2 reduced the migration potential of VCaP cells in wound healing assay and inhibited anchorage independent growth of these cells when compared to untreated cells. Annexin-V and TUNEL assays confirmed that VK2 induces apoptosis in VCaP cells. Our results also suggested that the VK2 has the ability to enforce growth arrest in CRPC cells by activating cellular senescence. Western blot analysis revealed that VK2 downregulated the expression of BiP, survivin, MMP-2, and PCNA while activating PARP-1, p21 and DNA damage response marker, phospho-H2AX in VCaP cells. Furthermore, VCaP cells treated with VK2 resulted in the activation of Caspase-3 and-7 apoptotic mediators. These results correlated with translocation of Bax and Cytochrome C to cytoplasm following VK2 treatment in VCaP cells as determined by confocal immunofluorescence analysis. In conclusion our study suggests that VK2 might be an effective anti-proliferative and anti-metastatic agent for CRPC by specifically targeting key anti-apoptotic, cell cycle progression and metastasis promoting signaling molecules. Citation Format: Subramanyam Dasari, Maarten C. Bosland, Andre Kajdacsy- Balla, Gnanasekar Munirathinam. Vitamin K2 targets castration-resistant prostate cancer VCaP cells by reactive oxygen species mediated apoptotic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 119. doi:10.1158/1538-7445.AM2017-119

Abstract 3305: Autophagic induction through upregulation of sestrin-2 transcription and mRNA stability mediates the anticancer effect of new compound ChlA-F

Abstract ChlA-F is a novel conformation-derivative of Cheliensisin A (Chel A), which is a new styryl-lactone isolated from Chinese Herb Goniothalamus cheliensis Huand shows potent antitumor potential effect both in vitro and in vivo. However, the anticancer activity and its potential mechanisms underlying ChlA-F action have never been explored. In present study, we evaluated the potency of ChlA-F on anchorage-independent growth and its association with autophagic responses in human high grade invasive bladder cancer cells. We found that ChlA-F significantly inhibited anchorage-independent growth of human bladder cancer cells by induction of autophagy in a sestrin2(SESN2)-dependent Fashion. Our results revealed that ChlA-F treatment specifically induced SESN2 expression via increasing its both gene transcription and mRNA stability. On the one hand, ChlA-F treatment markedly attenuated Dicer protein abundance, and in turn abolished miR-27a maturation and further relieved miR-27a directly binding to SESN2 mRNA 3’UTR, thereby promoting SESN2 mRNA stabilization. On the other hand,ChlA-F treatment promoted Sp1 abundance and consequently mediated SESN2 transcription. Moreover, either knockdown of Sp-1 or ectopic expression of miR-27a could abolish the autophagic response and reversed the inhibition of anchorage-independent growth in human bladder cancer cells. These results demonstrate that anticancer effect of ChlA-F is mediated by its activating autophagic pathway through promoting both SESN2 mRNA transcription and stabilization,which offers novel insights into the anticancer effect of ChlA-F on bladder cancer and provides a therapeutic alternative against human bladder cancer. Citation Format: Xiaohui Hua, Chuanshu Huang, Xueru Wu, Jingxia Li. Autophagic induction through upregulation of sestrin-2 transcription and mRNA stability mediates the anticancer effect of new compound ChlA-F [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3305. doi:10.1158/1538-7445.AM2017-3305

Role of STAT3 and FOXO1 in the Divergent Therapeutic Responses of Non-metastatic and Metastatic Bladder Cancer Cells to miR-145.

Although miR-145 is the most frequently downregulated miRNA in bladder cancer, its exact stage association and downstream effector have not been defined. Here, we found that miR-145 was upregulated in human patients with bladder cancer with lymph node metastasis and in metastatic T24T cell line. Forced expression of miR-145 promoted anchorage-independent growth of T24T cells accompanied by the downregulation of forkhead box class O1 (FOXO1). In contrast, in non-metastatic T24 cells, miR-145 overexpression inhibited cell growth with upregulation of FOXO1, and the knockdown of FOXO1 abolished the miR-145-mediated inhibition of cell growth. Mechanistic studies revealed that miR-145 directly bound to and attenuated 3'-untranslated region (UTR) activity of foxo1 mRNA in both T24 and T24T cells. Interestingly, miR-145 suppressed STAT3 phosphorylation at Tyr705 and increased foxo1 promoter transcriptional activity in T24 cells, but not in T24T cells, suggesting a role of STAT3 in the divergent responses to miR-145. Supporting this was our finding that STAT3 knockdown mimicked miR-145-mediated upregulation of FOXO1 in T24T cells and inhibition of anchorage-independent growth. Consistently, ectopic expression of miR-145 promoted tumor formation of xenograft T24T cells, whereas such promoting effect became inhibitory due to specific knockdown of STAT3. Together, our findings demonstrate the stage-specific association and function of miR-145 in bladder cancers and provide novel insights into the therapeutic targeting of miR-145. Mol Cancer Ther; 16(5); 924-35. ©2017 AACR.