Drug-induced Cell Cycle Arrest Research Articles

PDZ-binding kinase aggravates pancreatic neuroendocrine neoplasm progression by activating the AKT/mTOR pathway.

The therapeutic effects of existing drug regimens against pancreatic neuroendocrine neoplasms (pNENs) remain limited, and identifying ideal therapeutic targets is warranted. PDZ binding kinase (PBK) may play an oncogenic role in most solid tumors. However, its function in pNEN remains unclear. In this study, pNEN samples and International Cancer Genome Consortium data were used to determine the clinical significance of PBK. Cell counting and CCK8 assays were used to assess cell proliferation. Flow cytometry was used to assess drug-induced apoptosis and cell cycle arrest. An in vivo PBK-targeting experiment was performed in mice bearing pNENs. Western blotting, quantitative PCR, and immunohistochemistry were performed to assess the molecular mechanisms. PBK was significantly upregulated in pNEN tissues compared with paracancerous tissues. Additionally, PBK was a poor prognostic factor for pNEN patients. PBK was found to promote the proliferation of pNEN cells by activating the AKT/mTOR pathway. Furthermore, PBK inhibition combined with everolimus treatment had enhanced antitumour effects on pNEN via inhibiting AKT/mTOR pathway and inducing G0/G1 phase cell cycle arrest. This study highlights that PBK plays an oncogenic role in and is a promising therapeutic target for pNEN.

GPR17 signaling activation by CHBC agonist induced cell death via modulation of MAPK pathway in glioblastoma

AimGlioblastoma multiforme (GBM) is the most common and aggressive primary adult brain tumor. GBM is characterized by a heterogeneous population of cells that are resistant to chemotherapy. Recently, we have synthesized CHBC, a novel indole derivative targeted to GBM biomarker G-protein-coupled receptor 17 and inhibitor of GBM cells. In this study, CHBC was further investigated to characterize the efficiency of this agonist at the molecular level and its underlying mechanism in GBM cell death induction. Materials and methodsThe effect of CHBC and TMZ was determined using time dependent inhibitor assay in glioblastoma cells, LN229 and SNB19. Drug induced cell cycle arrest was measured using PI staining followed by image analysis. The induction of apoptosis and mechanism of action of CHBC was studied using apoptosis, caspase 3/7 and mitochondrial membrane permeability assays. Modulation of the key genes involved in MAPK signaling pathway was also measured using immunoblotting array. Key findingsThe inhibitory kinetic study has revealed that CHBC inhibited SNB19 and LN229 cell growth in a time-dependent manner. Furthermore, CHBC with the IC50 of 85 μM, mediated cell death through an apoptosis mechanism in both studied cell lines. The study also has revealed that CHBC targets GPR17 leading to the induction of apoptosis via the activation of Caspase 3/7 and dysfunction of mitochondrial membrane potential. In addition, CHBC treatment led to marked G2/M cell cycle arrest. The protein array has confirmed the anticancer effect of CHBC by the disruption of the mitogen-activated protein kinase pathway (MAPK). SignificanceTaken together, these results demonstrated that CHBC induced G2/M cell cycle arrest and apoptosis by disrupting MAPK signaling in human glioblastoma cells. This study concludes that CHBC represent a class of compounds for treating glioblastoma.

α-Mangostin and Doxorubicin Combination Synergistically Inhibited Cell Growth, Induced Cell Apoptosis with Increased Bak Protein and Decreased FLT3-ITD Phosphorylation in AML MOLM-13 Cell Line

Acute myeloid leukemia (AML) is associated with numerous mutations, with the Feline McDonough Sarcoma (FMS) like tyrosine kinase 3 (FLT3) mutation resulting in with poor prognosis and outcome. Therapies have been developed using FLT3 inhibitors, however, drug resistance often leads to disease relapse. In this study, α-Mangostin and doxorubicin (Dox) were evaluated, singly and in combination, for their anti-leukemic effect on MOLM-13, an AML cell line with FLT3-ITD mutation. Cell viability and apoptosis were determined using CyQUANTGR and TUNEL assay, respectively. Cell cycle analysis was conducted on propidium iodide-stained cells using flow cytometry. Cellular proteins were quantified using Western blot technique, with additional study by ELISA for FLT3 kinase activity. The results revealed that cell treatment by the combined drug, Dox (1 µM) and α-Mangostin (20 µM), compared to Dox (1 µM) alone, caused a significant inhibitory effect (P<0.001) and indicated synergistic cell growth inhibition. The combined drug also showed increased TUNEL positive apoptotic cells and increased expression of the pro-apoptotic protein Bak compared to Dox alone (P<0.05). Dox treated cells, as well as the combined drug induced cell cycle arrest at G2/M phase compared to untreated cells (P<0.05 and P<0.001, respectively). There was also statistically significant (P<0.05) reduction of cdc25 phosphatases (enzymes which play an important role in G2/M transition) by the combination drug compared to sole cell treatment by Dox. Furthermore, phosphorylated FLT3 protein expression was reduced when the combined treatment was compared to Dox only after 2 h (P<0.05) and after 24 h (P<0.001). Thus, Dox and α-Mangostin combined treatment inhibited FLT3 phosphorylation in MOLM-13 cells which could have contributed to G2M cell arrest and apoptosis via cdc25s and Bak proteins respectively. Further studies are warranted to further evaluate the potential of Dox and α-Mangostin combined drug as inhibitors of FLT3-ITD phosphorylation and its potential clinical relevance in AML treatment.

DDRE-30. HIGH-THROUGHPUT DRUG SCREENING OF FDA-APPROVED ANTINEOPLASTIC DRUGS FOR THE TREATMENT OF AGGRESSIVE MENINGIOMAS

Abstract The management of aggressive meningiomas remains challenging due to limited treatment options besides surgical removal and radiotherapy. High recurrence rates and lack of effective chemotherapies may be reasons for the unfavorable prognosis of these patients. Consequently, there is an urgent need to identify effective therapeutic agents. For this purpose, we performed a high-throughput screening utilizing a drug library consisting of 147 FDA-approved antineoplastic drugs on the anaplastic meningioma cell line NCH93 using CellTiter-Glo (Promega). Based on the lowest IC50, the top 5 drugs were selected including Bortezomib, Carfilzomib, Omacetaxine, Ixabepilone, and Romidepsin. Validation of candidate compounds was performed in Ben-Men-1 (grade I), NCH93 (grade III), and IOMM-Lee cells (grade III) using crystal violet assay. Dose-curve analysis revealed IC50 values in the lower nanomolar range for all compounds and all cell lines (0.2 – 16.2 nM). To further substantiate our findings, cell proliferation assessed by manual counting was significantly reduced by up to 90% by each candidate drug at 10xIC50 after 48 h (P < 0.001). Furthermore, cell migration was inhibited up to 60% by all candidate drugs at the respective IC50 (P < 0.05). However, colony formation was only significantly reduced by Bortezomib and Carfilzomib (P < 0.001). The impact of the drugs on cell cycle and apoptosis was analyzed by flow cytometry using Annexin V/PI staining. All candidate drugs induced cell cycle arrest at G0/G1 or G2/M phase (P < 0.001) and subsequently induced apoptosis. Among them, Bortezomib exhibited the most pronounced effect by up to 80% of apoptotic cells (P < 0.001). In summary, by utilizing a high-throughput drug screening we were able to identify Bortezomib, Carfilzomib, Omacetaxine, Ixabepilone, and Romidepsin as potent antineoplastic agents for the treatment of aggressive meningiomas.

Receptor-Driven ERK Pulses Reconfigure MAPK Signaling and Enable Persistence of Drug-Adapted BRAF-Mutant Melanoma Cells.

Targeted inhibition of oncogenic pathways can be highly effective in halting the rapid growth of tumors but often leads to the emergence of slowly dividing persister cells, which constitute a reservoir for the selection of drug-resistant clones. In BRAFV600E melanomas, RAF and MEK inhibitors efficiently block oncogenic signaling, but persister cells emerge. Here, we show that persister cells escape drug-induced cell-cycle arrest via brief, sporadic ERK pulses generated by transmembrane receptors and growth factors operating in an autocrine/paracrine manner. Quantitative proteomics and computational modeling show that ERK pulsing is enabled by rewiring of mitogen-activated protein kinase (MAPK) signaling: from an oncogenic BRAFV600E monomer-driven configuration that is drug sensitive to a receptor-driven configuration that involves Ras-GTP and RAF dimers and is highly resistant to RAF and MEK inhibitors. Altogether, this work shows that pulsatile MAPK activation by factors in the microenvironment generates a persistent population of melanoma cells that rewires MAPK signaling to sustain non-genetic drug resistance.

AICAR and Decitabine Enhance the Sensitivity of K562 Cells to Imatinib by Promoting Mitochondrial Activity.

Although the advent of tyrosine kinase inhibitors (TKIs) has dramatically improved the survival of patients with chronic myeloid leukaemia (CML), acquired drug resistance and TKI-insensitive leukaemic stem cells (LSCs) remain major obstacles to a CML cure. In recent years, the reprogramming of mitochondrial metabolism has emerged as a hallmark of cancers, including CML, and in turn may be exploited for therapeutic purposes. Here, we investigated the effects of several drugs on the mitochondrial function of the CML cell line K562 and found that 5-aminoimidazole-4-carboxamide ribotide (AICAR) and decitabine could effectively increase the ATP content and mitochondrial biogenesis. In addition, these two drugs induced cell cycle arrest and a decrease in colony-forming capacity and promoted K562 cell differentiation. Moreover, we demonstrated that treatment with AICAR or decitabine enhanced the sensitivity of K562 cells to imatinib, as evidenced by a combination treatment assay. Altogether, our findings indicate that TKIs combined with mitochondrial regulation may provide a therapeutic strategy for the treatment of CML.

Abstract 6235: BCL2 blockade in refractory and relapsed classic Hodgkin Lymphoma; Venetoclax sensitize cells to first line treatment

Abstract Classic Hodgkin Lymphoma (cHL) is a germinal center derived lymphoma with 9,000 new cases diagnosed each year in the US. Despite 70-80% early stage cHL patients respond to current systemic treatment, 10% of patients are refractory to first-line therapy and 30-40% relapse. Refractory and relapsed disease is currently the challenge when treating cHL patients. There are no biomarkers suitable to identify patients that would be refractory or that could relapse at the primary diagnosis. Furthermore, there is no specific therapy rather than rescue chemotherapy schemes, which fails in 50% of the cases and associates with high risk severe toxicity. This highlights the need to deeper understand the cHL molecular biology and the screening for prognosis biomarkers and therapeutic innovative approaches. We have previously reported that the alternative NFkB pathway, mediated by Rel-B and NIK (NFkB Inducing Kinase), plays an important role in cHL survival. Its constitutive activation sustains high BCL2 expression. We analyzed NIK and BCL2 citoplasm expression in Hodgkin Reed-Sternberg cells (HRS) in the lymph node biopsies of 112 cHL naïve of therapy patients by immunohistochemistry [51 female Md age and (range) 36 (6-88), 61 male 40.7 (9-78)]. The median follow-up period was 512, months (6 to 136 months). The univariate analysis showed no correlation between NIK or BCL2 expression and the prognosis clinical and pathological parameters, including the PET Scan indicated at the end of the first-line treatment, neither the molecular markers routinely assayed. The statistical significance was maintained in multivariate analysis (Logistic and Cox Regression p=0.01). The survival analysis, using the Kaplan-Meir curves, showed that patients with ≥60% positive HRS cells had a shorter disease-free survival (DFS) [Log Rank Test (Mantel Cox) p=0.002] and a reduced overall survival (OS) [Log Rank Test (Mantel Cox) p=0.02]. NIK expression did not correlate with prognosis. L1236, U-H01, KM-H2, SUPDH1 and L540 human cHL cell lines that express BCL2 protein, were sensitive to venetoclax a specific BCL2 inhibitor. The drug induced cell cycle arrest in S-Phase and G2-M when treated with 1uM each 24 hours during 10 days, as compared to wild type cells and cells treated with the vehicle. Furthermore, venetoclax sensitized cHL cell lines to vincristine and doxorubicin, two drugs used in the first-line chemotherapy scheme ABVD (Adriamycin, Bleomycin, vincristine, doxorubicin). In summary, we found that the alternative NFkB pathway plays an important role in the refractory and relapsed Hodgkin disease biology, being BCL2 a key downstream target. BCL2 performed well as a prognosis biomarker identifying refractory patients and those that relapsed being assayed at diagnosis of the primary disease. We believe BCL2 directed-therapy in cHL could be interested in patients that express this protein in ≥60% HRS cells in the lymph node biopsy performed at diagnosis. Furthermore venetoclax sensitized cHL cells to conventional chemotherapy. Citation Format: Angélica M. Cedeño-Gamboa, Mariángeles Díaz, Victoria Otero, Natalia Schutz, Dorotea Fantl, Federico Jauk, Myriam Nuñez, Stella M. Ranuncolo. BCL2 blockade in refractory and relapsed classic Hodgkin Lymphoma; Venetoclax sensitize cells to first line treatment [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 6235.

A Theoretical Model for the Cell Cycle and Drug Induced Cell Cycle Arrest of FUCCI Systems with Cell-to-Cell Variation during Mitosis.

Since the molecular mechanism of the cell cycle was established, various theoretical models of this process have been developed. A recent study revealed significant variability in cell cycle duration between mother and daughter cells, but this observation has not been incorporated into the theoretical models. We used fluorescent ubiquitination-based cell cycle indicator (FUCCI) systems and live-monitored the heterogeneity of cell cycle progression within daughter cells, which accounts for dephasing synchrony. To incorporate the variable cell cycle durations into a model, we modified a two-ordinary differential equation (ODE) model based on reciprocal activation between CDK1 and APC. Our model reproduced the experimental population profile, in which cell cycle synchrony dephased due to variability. Based on this model, we determined parameters for CDK1 and APC in the cell cycle profile after treatment with antimitotic drugs and associated the parameters with the drugs' mode of action as cell cycle inhibitors. This suggests that this model is useful for determining the mode of action of unknown small molecules on the cell cycle.

Oral Recombinant Methioninase Combined with Caffeine and Doxorubicin Induced Regression of a Doxorubicin-resistant Synovial Sarcoma in a PDOX Mouse Model.

Synovial sarcoma (SS) is a recalcitrant neoplasm with low chemosensitivity. We recently reported that recombinant methioninase (rMETase) inhibited SS growth in a patient-derived orthotopic xenograft (PDOX) mouse model and was more effective when administered in combination with the first-line drug doxorubicin (DOX). Caffeine enhances the efficacy of anticancer drugs by overcoming drug-induced cell-cycle arrest and increasing subsequent apoptosis. Here, we determined the efficacy of oral recombinant methioninase (o-rMETase) in combined with caffeine on an SS-PDOX model. Mice bearing SS-PDOX tumors were randomized into four treatment groups of six: Untreated control; o-rMETase alone; o-rMETase with caffeine; DOX plus o-rMETase with caffeine. Tumor size and body weight were measured during the treatment and plasma L-methionine (MET) levels were measured at the end of treatment. All treatments significantly inhibited SS-PDOX tumor growth. Combining caffeine with o-rMETase was more effective than o-rMETase alone. DOX combined with o-rMETase and caffeine led to regression of SS-PDOX. Plasma MET levels were reduced with o-rMETase treatment. These results suggest that combining o-rMETase and caffeine along with first-line chemotherapy can be highly effective for SS and has clinical potential for this recalcitrant disease.

The long non-coding RNA BC200 (BCYRN1) is critical for cancer cell survival and proliferation

BackgroundBC200 is a long non-coding RNA expressed at high levels in the brain and elevated in a variety of tumour types. BC200 has a hypothesized role in translational regulation; however, to date the functional role of BC200 in both normal and diseased states remains poorly characterized.MethodsDetailed BC200 expression analyses were performed in tumor cell lines, primary and non-tumorigenic cultured breast and lung cells, and a panel of normal human tissues by quantitative real-time PCR and confirmed by northern blot. Subcellular fractionation was performed to assess BC200 distribution and efficient knock-down of BC200 was established using both locked nucleic acid (LNA) GapmeRs and conventional siRNAs. Cell viability following BC200 knockdown and overexpression was assessed by MTT assay and induction of apoptosis was monitored by Annexin V/PI staining and flow cytometry. Cell cycle arrest and synchronization were performed using serum withdrawal as well as the specific inhibitors Lovastatin, Thymidine, RO3306 and Nocodazole. Synchronization was monitored by fluorescent analysis of cellular DNA content by flow cytometryResultsBC200 expression was substantially upregulated in brain and elevated expression was also observed in testes, small intestine and ovary. Expression in cultured tumour cells was dramatically higher than corresponding normal tissue; however, expression in cultured primary cells was similar to that in immortalized and cancer cell lines. BC200 knockdown resulted in a dramatic loss of viability through growth arrest and induction of apoptosis that could be partially rescued by overexpression of wild-type BC200 but not an siRNA-resistant sequence mutant. A substantial decrease in BC200 expression was observed upon cell confluence or serum deprivation, as well as drug induced cell cycle arrest in G1 or G2 but not S- or M-phases. Upon release from cell cycle arrest, BC200 expression was recovered as cells entered S-phase, but did not follow a periodic expression pattern during synchronized progression through the cell cycle. This elevated expression was critical for the survival of proliferating cancerous and non-cancerous cells, but is dispensable upon senescence or cell cycle arrest.ConclusionsBC200 expression is elevated in proliferating cultured cells regardless of origin. In primary cells, expression is dramatically reduced upon cell cycle arrest by confluence, serum deprivation or chemical inhibition. The lethality of BC200 knockdown is restricted to actively proliferating cells, making it a promising therapeutic target for a broad spectrum of cancers.

Abstract P3-03-01: A novel high-throughput secreted factor screen and bioinformatics pipeline identifies microenvironment-derived FGF2 as a mechanism of resistance to anti-estrogens, PI3K, and mTOR inhibitors in ER+ breast cancer

Abstract Resistance to anti-estrogen therapy is a serious obstacle to the treatment of patients with ER+ breast cancer. Unlike the majority of preclinical studies that focus on cancer cell-intrinsic mechanisms of resistance, we hypothesized that the tumor microenvironment significantly contributes to drug resistance, and that individual factors present in the microenvironment differentially modulate response to therapy. To systematically test this hypothesis, high-throughput screens were performed in which 297 unique secreted proteins (cytokines, growth factors, and extracellular matrix) were tested on ER+ breast cancer cell lines (MCF-7 and T47D) treated +/- anti-estrogen fulvestrant or the PI3K inhibitor GDC-0941 (pictilisib). Cytokines that rescued cells (hits) were validated in two additional ER+ breast cancer cell lines (ZR75-1 and HCC-1500), and expanded to include the anti-estrogen tamoxifen and the mTORC1 inhibitor everolimus. Hits were also found to rescue from combinations of fulvestrant/GDC-0941 and fulvestrant/everolimus in models with acquired resistance to fulvestrant. Multiple factors, such as ErbB family ligands and fibroblast growth factors (FGFs), were among the top validated hits. To parse out which hits are most likely to be relevant in the setting of the ER+ breast tumor microenvironment, a bioinformatics filter was developed to incorporate gene and protein expression data for secreted proteins in non-cancer human tissues relevant to ER+ breast cancer. These tissues include primary breast tissue components (breast mammary, adipose, primary fibroblasts) and common metastatic sites (bone marrow, lung, liver). After filtering, the top hit was fibroblast growth factor 2 (FGF2), which significantly drives resistance to anti-estrogens, PI3K, and mTOR inhibitors, and is highly expressed in non-cancer tissues relevant to ER+ breast cancer. FGF2-mediated rescue was completely abrogated by the ATP-competitive pan-FGFR inhibitor PD173074, confirming FGFR kinase specificity of the rescue phenotype. Immunoblot data confirmed that FGF2 induced phosphorylation of FGFR and the FGFR effector FRS2, and a consistent pattern of downstream MEK-ERK-Rsk90 activation for rescue to all drugs. Flow cytometry data showed that FGF2-mediated rescue leads to decreases in drug-induced apoptosis and G1 cell cycle arrest, which correspond at the protein level to increased degradation of the pro-apoptotic protein Bim and upregulation of the cell cycle driver cyclin D1, respectively. Mice bearing MCF-7 xenografts were treated with vehicle, FGF2 (20 ug/kg/d, s.c.), fulvestrant (5 mg/wk, s.c.), or the combination. Vehicle-treated and FGF2-treated mice showed similar rates of tumor growth. Single-agent fulvestrant significantly suppressed tumor growth, while the addition of FGF2 rescued from the growth-inhibitory effect of fulvestrant. Studies in other tumor models are ongoing. These data collectively suggest that stroma-derived FGF2-mediated drug resistance is a novel therapeutic opportunity in ER+ breast cancer. Citation Format: Shee K, Hinds JW, Hampsch RA, Golub TR, Straussman R, Miller TW. A novel high-throughput secreted factor screen and bioinformatics pipeline identifies microenvironment-derived FGF2 as a mechanism of resistance to anti-estrogens, PI3K, and mTOR inhibitors in ER+ breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-03-01.

Abstract 3795: Cardiac glycoside digitoxin and its monosaccharide analogue MonoD inhibit NFκB to induce apoptotic cell death in ER+ MCF7 and triple-negative MDAMB-468 breast cancer cells

Abstract Digitoxin, a cardiac glycoside well appreciated in the treatment of congestive heart failure, has been poorly understood and appreciated as an anti-cancer therapeutic due to its narrow therapeutic potential. Using a patented palladium-catalyzed de novo synthesis method, we have developed a library of digitoxin analogs, which alleviate the toxic side effects of digitoxin while maintaining its chemical activity. We have evaluated MonoD, a digitoxin analog with a single sugar moiety, in a variety of NCI-60 panel of cell lines, with a significant tumoristatic effect observed in estrogen receptor positive and triple negative breast cancer cells. Therefore, in order to determine the efficacy of digitoxin and MonoD, delineate its mode of action and identify molecular targets we chose to test this novel compound in ER+ MCF7 and TN MDAMB-468 breast cancer cells. Digitoxin and MonoD showed a dose dependent decrease in cell viability in MCF7 and MDAMB-468 cell lines. Further characterization of cell death using the Hoechst assay showed that MonoD induced significantly higher apoptosis at the same dose as digitoxin. Both drugs induce oxidative stress in the form of superoxide as assayed using Dihydroethidium (DHE). In vitro scratch migration assay showed a five-fold decrease in rate of cell migration in the presence of test drugs. MCF7 and MDAMB-468 cells were analyzed for cell cycle progression in the presence of digitoxin and MonoD. We observed that the drugs induced cell cycle arrest in G0/G1 phase at the expense of S and G2/M phases. Increased expression of p27Kip1 in the presence of drugs provided further validation, as increased p27 expression is known to block cell cycle progression through G1-S transition. In addition to cell cycle arrest, apoptosis is another widely accepted mechanism by which anti-cancer drugs inhibit tumor formation. We observed that these drugs induced apoptosis as indicated by cleaved PARP and cleaved caspase-9, in addition to down-regulation of pro-survival factor Bcl-2. NFκB has been reported to be constitutively active in many types of cancer by controlling the expression of several genes involved in cellular proliferation and apoptosis. We observed that NFκB expression was down-regulated in both cell lines with digitoxin and MonoD treatment. IKKβ, a critical kinase involved in regulating NFκB pathway showed significant down-regulation, thereby suggesting that these drugs exert their anti-cancer therapeutic potential through the inhibition of NFκB pathway. While additional work is required to establish their mode of action, we have sufficient evidence to hypothesize that down-regulation of NFκB is critical to the mode of action of these drugs. Further, it is evident from densitometric analyses of western blots, MTT and Hoechst assays that MonoD shows higher efficacy as compared to digitoxin in inducing cancer cell death. Citation Format: Yogesh Kulkarni, Vivek Kaushik, Raj Venkatadri, Clayton Wright, Juan Sebastian Yakisich, George O'Doherty, Neelam Azad, Anand Iyer. Cardiac glycoside digitoxin and its monosaccharide analogue MonoD inhibit NFκB to induce apoptotic cell death in ER+ MCF7 and triple-negative MDAMB-468 breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3795. doi:10.1158/1538-7445.AM2015-3795

Effect of poly(ADP-ribose)polymerase and DNA topoisomerase I inhibitors on the p53/p63-dependent survival of carcinoma cells

Depending on their genetic background (p53wt versus p53null), carcinoma cells are more or less sensitive to drug-induced cell cycle arrest and/or apoptosis. Among the members of the p53 family, p63 is characterized by two N-terminal isoforms, TAp63 and ΔNp63. TAp63 isoform has p53-like functions, while ΔNp63 acts as a dominant negative inhibitor of p53. We have previously published that TAp63 is involved in poly(ADP-ribose)polymerase-1 (PARP-1) signaling of DNA damage deriving from DNA topoisomerase I (TOP I) inhibition in carcinoma cells.In the present study, we treated MCF7 breast carcinoma cells (p53+/ΔNp63−) or SCC022 (p53−/ΔNp63+) squamous carcinoma cells with the TOP I inhibitor topotecan (TPT) and the PJ34 PARP inhibitor, to compare their effects in the two different cell contexts.In MCF7 cells, we found that PJ34 addition reverts TPT-dependent PARP-1 auto-modification and triggers caspase-dependent PARP-1 proteolysis. Moreover, TPT as single agent stimulates p53ser15 phosphorylation, p53 PARylation and occupancy of the p21WAF promoter by p53 resulting in an increase of p21WAF expression. Interestingly, PJ34 in combination with TPT enhances p53 occupancy at the BAX promoter and is associated with increased BAX protein level.In SCC022 cells, instead, TPT+PJ34 combined treatment reduces the level of the anti-apoptotic ΔNp63α protein without inducing apoptosis. Remarkably, in such cells, either exogenous p53 or TAp63 can rescue the apoptotic program in response to the treatment.All together our results suggest that in cancer cells PARP inhibitor(s) can operate in the choice between growth arrest and apoptosis by modulating p53 family-dependent signal

Digital holographic microscopy for non-invasive monitoring of cell cycle arrest in L929 cells.

Digital holographic microscopy (DHM) has emerged as a powerful non-invasive tool for cell analysis. It has the capacity to analyse multiple parameters simultaneously, such as cell- number, confluence and phase volume. This is done while cells are still adhered and growing in their culture flask. The aim of this study was to investigate whether DHM was able to monitor drug-induced cell cycle arrest in cultured cells and thus provide a non-disruptive alternative to flow cytometry. DHM parameters from G1 and G2/M cell cycle arrested L929 mouse fibroblast cells were collected. Cell cycle arrest was verified with flow cytometry. This study shows that DHM is able to monitor phase volume changes corresponding to either a G1 or G2/M cell cycle arrest. G1-phase arrest with staurosporine correlated with a decrease in the average cell phase volume and G2/M-phase arrest with colcemid and etoposide correlated with an increase in the average cell phase volume. Importantly, DHM analysis of average cell phase volume was of comparable accuracy to flow cytometric measurement of cell cycle phase distribution as recorded following dose-dependent treatment with etoposide. Average cell phase volume changes in response to treatment with cell cycle arresting compounds could therefore be used as a DHM marker for monitoring cell cycle arrest in cultured mammalian cells.

Targeting phospho-MARCKS overcomes drug-resistance and induces antitumor activity in preclinical models of multiple myeloma.

Multiple myeloma (MM) is incurable in virtually all patients due to the presence of innate and emergent drug-resistance. To identify potential drug resistance mechanisms in MM we used iTRAQ (isobaric tags for relative and absolute quantitation) mass spectrometry to compare protein expression profiles of drug-resistant (RPMI 8226-R5) and sensitive (RPMI 8226-S) isogenic cell lines. We identified selective overexpression of myristoylated alanine-rich C-kinase substrate (MARCKS) in drug-resistant R5 cells. MARCKS overexpression was also observed in several drug-resistant human myeloma cell lines (HMCLs) and in drug-resistant primary MM samples. Functionally, inhibition of MARCKS phosphorylation by enzastaurin or knockdown of the gene by RNAi significantly enhanced the sensitivity of resistant HMCLs and primary MM samples to bortezomib and to other anti-myeloma drugs, providing evidence that MARCKS can modulate drug response. Mechanistically, pMARCKS (phosphorylated form of MARCKS) was found to function as an E2F-1 cofactor to regulate SKP2 transcription. pMARCKS promoted cell-cycle progression by facilitating SKP2 expression, suppressing p27(Kip1) and potentially counteracting drug-induced cell-cycle arrest by promoting Cyclin E/CDK2 activity. Importantly, MARCKS knockdown in combination with bortezomib treatment overcame bortezomib resistance, significantly inhibited tumor growth and prolonged host survival in a MM xenograft model. These data provide a rationale for therapeutic targeting of pMARCKS to improve the outcome of patients with refractory/relapsed MM.

Dietary lutein modulates growth and survival genes in prostate cancer cells.

Lutein is a carotenoid pigment present in fruits and vegetables that has anti-inflammatory and antitumor properties. In this study, we examined the effect of lutein on proliferation and survival-associated genes in prostate cancer (PC-3) cells. We found that in vitro culture of PC-3 cells with lutein induced mild decrease in proliferation that improved in combination treatment with peroxisome proliferator-activated receptor gamma (PPARγ) agonists and other chemotherapeutic agents. Flow cytometry analyses showed that lutein improved drug-induced cell cycle arrest and apoptosis in prostate cancer. Gene array and quantitative reverse transcription-polymerase chain reaction analyses showed that lutein altered the expression of growth and apoptosis-associated biomarker genes in PC-3 cells. These findings highlight that lutein modulates the expression of growth and survival-associated genes in prostate cancer cells.

LIMK2 Mediates Resistance to Chemotherapeutic Drugs in Neuroblastoma Cells through Regulation of Drug-Induced Cell Cycle Arrest

Drug resistance is a major obstacle for the successful treatment of many malignancies, including neuroblastoma, the most common extracranial solid tumor in childhood. Therefore, current attempts to improve the survival of neuroblastoma patients, as well as those with other cancers, largely depend on strategies to counter cancer cell drug resistance; hence, it is critical to understand the molecular mechanisms that mediate resistance to chemotherapeutics. The levels of LIM-kinase 2 (LIMK2) are increased in neuroblastoma cells selected for their resistance to microtubule-targeted drugs, suggesting that LIMK2 might be a possible target to overcome drug resistance. Here, we report that depletion of LIMK2 sensitizes SHEP neuroblastoma cells to several microtubule-targeted drugs, and that this increased sensitivity correlates with enhanced cell cycle arrest and apoptosis. Furthermore, we show that LIMK2 modulates microtubule acetylation and the levels of tubulin Polymerization Promoting Protein 1 (TPPP1), suggesting that LIMK2 may participate in the mitotic block induced by microtubule-targeted drugs through regulation of the microtubule network. Moreover, LIMK2-depleted cells also show an increased sensitivity to certain DNA-damage agents, suggesting that LIMK2 might act as a general pro-survival factor. Our results highlight the exciting possibility of combining specific LIMK2 inhibitors with anticancer drugs in the treatment of multi-drug resistant cancers.

Enhanced anti‐tumour effects of Vinca alkaloids given separately from cytostatic therapies

In polychemotherapy protocols, that is for treatment of neuroblastoma and Ewing sarcoma, Vinca alkaloids and cell cycle-arresting drugs are usually administered on the same day. Here we studied whether this combination enables the optimal antitumour effects of Vinca alkaloids to be manifested. Vinca alkaloids were tested in a preclinical mouse model in vivo and in vitro in combination with cell cycle-arresting drugs. Signalling pathways were characterized using RNA interference. In vitro, knockdown of cyclins significantly inhibited vincristine-induced cell death indicating, in accordance with previous findings, Vinca alkaloids require active cell cycling and M-phase transition for induction of cell death. In contrast, anthracyclines, irradiation and dexamethasone arrested the cell cycle and acted like cytostatic drugs. The combination of Vinca alkaloids with cytostatic therapeutics resulted in diminished cell death in 31 of 36 (86%) tumour cell lines. In a preclinical tumour model, anthracyclines significantly inhibited the antitumour effect of Vinca alkaloids in vivo. Antitumour effects of Vinca alkaloids in the presence of cytostatic drugs were restored by caffeine, which maintained active cell cycling, or by knockdown of p53, which prevented drug-induced cell cycle arrest. Therapeutically most important, optimal antitumour effects were obtained in vivo upon separating the application of Vinca alkaloids from cytostatic therapeutics. Clinical trials are required to prove whether Vinca alkaloids act more efficiently in cancer patients if they are applied uncoupled from cytostatic therapies. On a conceptual level, our data suggest the implementation of polychemotherapy protocols based on molecular mechanisms of drug-drug interactions. This article is commented on by Solary, pp 1555-1557 of this issue. To view this commentary visit http://dx.doi.org/10.1111/bph.12101.

The mitochondrial transport protein SLC25A43 affects drug efficacy and drug-induced cell cycle arrest in breast cancer cell lines

The mitochondria have been identified as key players of apoptosis, cell proliferation and cell cycle regulation. However, the role of mitochondria in breast cancer and treatment failure remains unclear. We have previously shown a common deletion of the gene SLC25A43 in human epidermal growth factor receptor 2 (HER2)-positive breast cancer. This gene is coding for a mitochondrial inner membrane transporter and, to date, little is known about the function of this protein. We have also found that low protein expression of SLC25A43 significantly correlates with a lower S phase fraction in HER2-positive breast cancer. The aim of this study was to investigate whether knockdown (KD) of SLC25A43 could have an effect on the cytotoxicity of different cytostatic drugs using MCF10A, MCF7 and BT-474 cells. Following siRNA-mediated KD of SLC25A43, one non-malignant and two breast cancer cell lines were exposed to the anthracycline epirubicin or the taxane paclitaxel. The HER2-positive breast cancer cells were also exposed to the targeted therapy trastuzumab and dual exposure to trastuzumab and paclitaxel. We found that KD of SLC25A43 resulted in a decreased cytotoxic effect of paclitaxel in the two cancer cell lines (P<0.05). Further analysis of cell cycle phase distribution showed that KD increased the paclitaxel-induced G2/M block in these two cell lines (P<0.05). KD of SLC25A43 also reduced the inhibitory effect of trastuzumab on cell proliferation in the HER2-positive cancer cell line BT-474 (P<0.05), and the drug-induced G0/G1 block (P<0.05). Moreover, SLC25A43 influenced the percentage of Ki-67-positive cells. Our findings demonstrate that the mitochondrial protein SLC25A43 affects drug efficacy and cell cycle regulation following drug exposure in breast cancer cell lines.

TRAIL Preferentially Affects Cell Cycle-Arrested Tumor Cells Including Stem- and Progenitor Cells From Patients with Acute Lymphoblastic Leukemia

AbstractAbstract 1879Leukemic stem- and progenitor cells exhibit low cycling activity which might represent a major cause for their increased treatment resistance. TRAIL (TNF-related apoptosis inducing ligand) is a novel putative anticancer drug currently in phase I and II clinical testing. We recently showed that TRAIL is able to address stem- and progenitor cells from patients with acute lymphoblastic leukemia (ALL) in xenotransplantation assays (Alves et al., Blood 2012,119,4224). As stem- and progenitor cells are often non-cycling, we asked here, whether TRAIL is able to address resting leukemia cells.We used cell lines and primary tumor cells from children with ALL which were amplified in severely immuno-compromised mice (NSG mice). Cell cycle arrest was induced (i) by addition of conventional cytotoxic drugs which are known to act as cytostatic drugs such as doxorubicine; (ii) by biochemical inhibitors known to induce cell cycle arrest at different defined points of the cell cycle such as mimosine; (iii) by molecular approaches and knockdown of cyclinB arresting cell cycle in G2 or knockdown of cyclinE arresting cell cycle in G1.Unexpectedly, TRAIL-induced apoptosis was enhanced, whenever cell cycle was arrested. Cell cycle arrest sensitized towards TRAIL-induced apoptosis independently from the point or phase of cell cycle which was arrested (G0, G1 or G2) and independently from the agent used to arrest the cell cycle. Similarly, knockdown of cyclinB or cyclinE both clearly sensitized cell line cells towards TRAIL-induced apoptosis.Cytotoxic drugs and cell cycle inhibitors might arrest the cell cycle by activation of p53. Accordingly, when caffeine was added which inhibited p53 activity and drug-induced cell cycle arrest, sensitization towards TRAIL-induced apoptosis was blocked.We have recently established a novel method which enables performing knockdown experiments in tumor cells derived from ALL patients (Höfig et al., Cell Comm. Signal. 2012,10,8). Using this method and most important for clinical translation, we could show that knockdown of either cyclinB or cyclinE clearly sensitized patient-derived ALL cells towards TRAIL-induced apoptosis.Taken together and in contrast to most conventional cytotoxic drugs, TRAIL exerts anti-tumor activity preferentially against tumor cells in cell cycle arrest and less against actively cycling tumor cells. This special feature of TRAIL might explain its anti-tumor activity against stem- and progenitor cells in patients with ALL. Thus, TRAIL might represent an interesting drug to treat disease stages with accumulation of stem- and progenitor cells and static tumor disease, e.g., during minimal residual disease. Disclosures:No relevant conflicts of interest to declare.