scholarly journals CD133 Promotes Resistance to Trametinib in Melanoma Stem Cells by Activating an AKT / BCL-2 Survival Pathway

2021 ◽  
Author(s):  
Cynthia Marie Simbulan-Rosenthal ◽  
Yogameenakshi Haribabu ◽  
Sahar Vakili ◽  
Li-Wei Kuo ◽  
Havens Clark ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Active Form ◽  
Family Members ◽  
Mek Inhibitor ◽  
Melanoma Cells ◽  
Parp Cleavage ◽  
Stem Cell Markers ◽  
Survival Pathway ◽  
Induced Apoptosis ◽  
Sirna Knockdown

Malignant melanoma is a lethal skin cancer containing melanoma-initiating cells (MIC), implicated in tumorigenesis, invasion, and drug resistance, and characterized by elevated expression of stem cell markers, such as CD133. We previously showed that siRNA knockdown of CD133 enhances apoptosis induced by the MEK inhibitor trametinib in melanoma cells. The current study investigates underlying mechanisms of CD133’s anti-apoptotic activity in patient-derived BAKP and POT cells, harboring difficult-to-treat NRASQ61K and NRASQ61R drivers, after CRISPR-Cas9 CD133 knockdown or Dox-inducible expression of CD133. To maintain stable expression of CD133, MACS-sorted CD133(+) positive cells were expanded by ROCK-mediated conditional reprogramming of BAKP melanoma cells (BAKR). BAKR showed increased survival via reduced apoptosis after exposure to trametinib or DTIC, compared to BAKP. CRISPR-Cas9- mediated CD133 knockdown in BAKR cells (BAKR-T3) re-sensitized the cells, while CRISPR-Cas9 knockdown of CD133 in parental BAKP and POT cells even further increased trametinib-induced apoptosis (cleaved PARP) by reducing levels of anti-apoptotic BCL-xL, p-AKT, and p-BAD, and increasing pro-apoptotic BAD and active BAX. Dox-induced CD133 overexpression had the opposite effect, and blocked trametinib-induced apoptosis in both cell lines, coincident with elevated p-AKT, p-BAD, BCL-2 and BCL-xL and decreased levels of the active form of BAX and caspases-3 and -9. The roles of CD133 in AKT and BAD phosphorylation, or in the upregulation of anti-apoptotic BCL-2 family members, was further investigated by AKT knockout with siRNA, or inhibition of BCL-2 family members with navitoclax (ABT-263). Similar to CD133 knockdown, AKT1/2 siRNA knockdown in BAKP cells also reduced p-BAD. CD133 knockdown (T3)-mediated reduction of pBAD levels was equivalent in AKT-knockdown or AKT control cells indicating that CD133 may be upstream of AKT signaling. In BAKP cells treated with trametinib and/or ABT-263, effects of ABT-263 mirrored CD133 knockdown, since levels of active BAX and cleaved-PARP in BAKP-SC (CD133-) cells increased to the same level as that exhibited by BAKP-T3 cells (CD133+). CD133 may therefore activate a survival pathway where 1) increased phosphorylation of AKT induces 2) phosphorylation and inactivation of BAD, 3) decrease in the active form of BAX, and 4) reduction in caspase-mediated PARP cleavage, indicating apoptosis suppression leading to drug resistance in melanomas. Targeting survival pathways by which CD133 may confer chemoresistance in MICs can contribute to development of more effective treatments for patients with high-risk melanoma.

scholarly journals CD133 Is Associated with Increased Melanoma Cell Survival after Multikinase Inhibition

2019 ◽  
Vol 2019 ◽  
pp. 1-19 ◽  
Author(s):  
Cynthia M. Simbulan-Rosenthal ◽  
Anirudh Gaur ◽  
Hengbo Zhou ◽  
Maryam AbdusSamad ◽  
Qing Qin ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Kinase Inhibitors ◽  
Braf Inhibitor ◽  
Mek Inhibitor ◽  
Melanoma Cells ◽  
Stem Cell Marker ◽  
High Dose ◽  
Recurrence Rates ◽  
Mapk Inhibitors

FDA-approved kinase inhibitors are now used for melanoma, including combinations of the MEK inhibitor trametinib, and BRAF inhibitor dabrafenib for BRAFV600 mutations. NRAS-mutated cell lines are also sensitive to MEK inhibitionin vitro, and NRAS-mutated tumors have also shown partial response to MEK inhibitors. However, melanoma still has high recurrence rates due to subpopulations, sometimes described as “melanoma initiating cells,” resistant to treatment. Since CD133 is a putative cancer stem cell marker for different cancers, associated with decreased survival, we examined resistance of patient-derived CD133(+) and CD133(-) melanoma cells to MAPK inhibitors. Human melanoma cells were exposed to increasing concentrations of trametinib and/or dabrafenib, either before or after separation into CD133(+) and CD133(-) subpopulations. In parental CD133-mixed lines, the percentages of CD133(+) cells increased significantly (p<0.05) after high-dose drug treatment. Presorted CD133(+) cells also exhibited significantly greater (p<0.05) IC50s for single and combination MAPKI treatment. siRNA knockdown revealed a causal relationship between CD133 and drug resistance. Microarray and qRT-PCR analyses revealed that ten of 18 ABC transporter genes were significantly (P<0.05) upregulated in the CD133(+) subpopulation, while inhibition of ABC activity increased sensitivity, suggesting a mechanism for increased drug resistance of CD133(+) cells.

Targeting Microenvironment-Mediated NFκb Activation With MLN4924, An Inhibitor Of The Nedd8-Activating Enzyme, In Chronic Lymphocytic Leukemia B Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2875-2875
Author(s):  
Claire Godbersen ◽  
Alan Eastman ◽  
Jennifer R. Brown ◽  
Alexey V. Danilov
Keyword(s):  
Cell Cycle ◽  
B Cells ◽  
Family Members ◽  
Ubiquitin Ligases ◽  
Parp Cleavage ◽  
Pathway Activity ◽  
Nfκb Pathway ◽  
Induced Apoptosis ◽  
Cells Cultured

Abstract Gene expression profiling identified lymph nodes as the site of cell activation and proliferation with upregulation of NFκB. B-cell receptor (BCR) and CD40 signaling enhances NFκB pathway activity in CLL cells resident in the microenvironment, leading to their survival, proliferation and chemoresistance. The NFκB pathway has been successfully targeted in CLL in vitro, yet lack of clinical advances with those agents necessitates development of novel approaches. Ubiquitination of IκBα may partially account for constitutive activation of NFκB in this setting. MLN4924 is an investigational agent that inhibits the Nedd8-activating enzyme and thereby prevents neddylation of Cullin-RING ubiquitin ligases, resulting in a decrease of their activity and stabilization of their protein substrates. This study reports that MLN4924 abrogates pro-survival microenvironment stimuli in CLL cells in vitro. Primary B cells from 50 patients with CLL were co-cultured in vitro with CD40L-expressing stroma to mimic the pro-survival conditions present in lymphoid tissue. The effect of MLN4924 on CLL cell apoptosis, NFκB pathway activity, Bcl-2 family members and cell cycle was assessed by flow cytometry, western blotting, PCR and immunocytochemistry. MLN4924 was provided by Millennium Pharmaceuticals. Treatment with MLN4924 led to reduced neddylation of Cullin-RING ubiquitin ligases, accumulation of phospho-IκBa and induced modest apoptosis in CLL cells cultured off stroma (12.8±1.7% after a 24 hour incubation with 1 μM MLN4924 compared to untreated control). Incubation with the CD40L-expressing stroma resulted in activation of the canonical and non-canonical NFκB pathways in the CLL cells, induction of Mcl-1 and Bcl-xL, leading to protection from spontaneous apoptosis. This was accompanied by repression of Bim, a pro-apoptotic BH3-only protein of the Bcl-2 family. CD40L-expressing stroma rescued CLL cell from drug-induced apoptosis, including bendamustine, fludarabine, chlorambucil and CAL-101. By contrast, CD40L-expressing stroma failed to elicit resistance to MLN4924. Moreover, CLL cells cultured on stroma were more sensitive to Nedd8-activating enzyme inhibition compared to cells off stroma (1 μM MLN4924 induced apoptosis in 55.3±3.4% cells cultured on stroma). MLN4924 promoted apoptosis regardless of the common adverse prognostic factors (IGHV mutational status, cytogenetic markers, or ZAP-70 expression). In the presence of MLN4924, CLL cells demonstrated a significant decrease in nuclear translocation of the NFκB subunits p65 and p52 thus marking de-activation of both canonical and non-canonical pathways. Using gene microarray analysis we determined that receptor signaling and expression target pathways involving NFκB were most significantly associated with genes downregulated in CLL cells by MLN4924 (p<0.0001). We noted reduced transcription of several groups of NFκB target genes, including anti-apoptotic Bcl-2 family members and genes involved in cell cycle progression (p<0.01). Furthermore, treatment with MLN4924 resulted in a significant downregulation of a number of important cytokine ligands and receptors expressed by the CLL cells, including CCL5, CCL22, CXCR7, CXCR5 and CD40. Concomitantly, MLN4924 promoted induction of Bim and Noxa in the CLL cells which preceded apoptosis as evidenced by PARP cleavage. siRNA-mediated knockdown of either Bim or Noxa resulted in partial protection from MLN4924-mediated apoptosis irrespective of expression of the pro-survival Bcl-2 family members (Bcl-2, Bcl-xL or Mcl-1). This indicates that MLN4924 leads to rebalancing of Bcl-2 family members towards the pro-apoptotic BH3-only proteins in CLL. Furthermore, MLN4924 reversed stroma-mediated protection from fludarabine and the alkylators. In summary, we were able to abrogate the protective effect of the CLL microenvironment in vitro using MLN4924, a Nedd8-activating enzyme inhibitor. MLN4924 reduced NFκB activity in CLL cells, led to re-expression of the pro-apoptotic BH3-only proteins and interfered the chemokine network thus carrying a potential to disrupt cell homing. These results suggest a rationale for clinical investigation of MLN4924 as a novel tool to target microenvironment in CLL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Molecular Targeting of HuR Oncoprotein Suppresses MITF and Induces Apoptosis in Melanoma Cells

Cancers ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 166
Author(s):  
Rebaz Ahmed ◽  
Ranganayaki Muralidharan ◽  
Akhil Srivastava ◽  
Sarah E. Johnston ◽  
Yan D. Zhao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Drug Resistance ◽  
Targeted Therapy ◽  
Antitumor Activity ◽  
Mek Inhibitor ◽  
Melanoma Cells ◽  
Migration And Invasion ◽  
Mutational Status ◽  
The Impact

Background: Treatment of metastatic melanoma possesses challenges due to drug resistance and metastases. Recent advances in targeted therapy and immunotherapy have shown clinical benefits in melanoma patients with increased survival. However, a subset of patients who initially respond to targeted therapy relapse and succumb to the disease. Therefore, efforts to identify new therapeutic targets are underway. Due to its role in stabilizing several oncoproteins’ mRNA, the human antigen R (HuR) has been shown as a promising molecular target for cancer therapy. However, little is known about its potential role in melanoma treatment. Methods: In this study, we tested the impact of siRNA-mediated gene silencing of HuR in human melanoma (MeWo, A375) and normal melanocyte cells in vitro. Cells were treated with HuR siRNA encapsulated in a lipid nanoparticle (NP) either alone or in combination with MEK inhibitor (U0126) and subjected to cell viability, cell-cycle, apoptosis, Western blotting, and cell migration and invasion assays. Cells that were untreated or treated with control siRNA-NP (C-NP) were included as controls. Results: HuR-NP treatment significantly reduced the expression of HuR and HuR-regulated oncoproteins, induced G1 cell cycle arrest, activated apoptosis signaling cascade, and mitigated melanoma cells’ aggressiveness while sparing normal melanocytes. Furthermore, we demonstrated that HuR-NP treatment significantly reduced the expression of the microphthalmia-associated transcription factor (MITF) in both MeWo and MITF-overexpressing MeWo cells (p < 0.05). Finally, combining HuR-NP with U0126 resulted in synergistic antitumor activity against MeWo cells (p < 0.01). Conclusion: HuR-NP exhibited antitumor activity in melanoma cells independent of their oncogenic B-RAF mutational status. Additionally, combinatorial therapy incorporating MEK inhibitor holds promise in overriding MITF-mediated drug resistance in melanoma.

SDX-101 Is Cytotoxic and Overcomes Drug Resistance in Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3466-3466
Author(s):  
Makoto Hamasaki ◽  
Teru Hideshima ◽  
Kenji Ishitsuka ◽  
Hiroshi Yasui ◽  
Norihiko Shiraishi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Antineoplastic Agent ◽  
Parp Cleavage ◽  
Family Protein ◽  
Dose Dependent Fashion ◽  
Peripheral Mononuclear Cells ◽  
Induced Apoptosis

Abstract SDX-101 is an oral antineoplastic agent, has been evaluated in a phase I/II study in B-cell malignancies. It induces cytotoxicity at least in part, by significantly inhibiting expression of Mcl-1, an anti-apoptotic Bcl-2 family protein which is highly expressed in CLL cells. Here, we examined the cytotoxicity of SDX-101 against multiple myeloma (MM) cell lines. SDX-101 significantly inhibited growth in MM.1S, U266, RPMI8226 MM cell lines using MTT assays in a time- and dose-dependent fashion, with IC50s of 0.6mM, 1.0mM, and 0.4mM, respectively. In contrast, SDX-101 did not induce cytotoxicity in normal peripheral mononuclear cells (PBMCs) at these concentrations. Importantly, SDX-101 induced cytotoxicity even in dexamethasone (MM.1R)-, doxorubicin (RPMI-Dox40)-, and melphalan (LR5)- resistant MM cell lines. SDX-101 (0.3–1.25mM) triggered apoptosis associated with pro-caspase-3, pro-caspase-8, and PARP cleavage, as confirmed by immunoblotting. Although, interleukin-6 (IL-6) and insulin-like growth factor (IGF)-1 completely abrogates Dex-induced MM cell apoptosis, neither protects against SDX-101-induced apoptosis in MM.1S and RPMI8226 cells. Moreover, dexamethasone, Melphalan, and AS2O3 augment apoptosis induced by SDX-101. Importantly, SDX-101 downregulated both b-catenin and cyclin D1 expression in RPMI8226 cells. Finally, our recent studies demonstrate that the bone marrow (BM) microenvironment promotes MM cell growth, survival, and drug resistance while SDX-101 inhibits viability even if MM cells adherent to BM stromal cells; an environment in which cytotoxic potency is lost with other anti-neoplastic agents. Our data therefore demonstrate that SDX-101 induces apoptosis in MM cells via a mechanism different from conventional MM drugs, and support clinical trials of this agent, alone or in combination with conventional and/or novel agents to improve therapeutic outcome in MM.

scholarly journals Diverse marrow stromal cells protect CLL cells from spontaneous and drug-induced apoptosis: development of a reliable and reproducible system to assess stromal cell adhesion-mediated drug resistance

Blood ◽  
2009 ◽  
Vol 114 (20) ◽  
pp. 4441-4450 ◽  
Author(s):  
Antonina V. Kurtova ◽  
Kumudha Balakrishnan ◽  
Rong Chen ◽  
Wei Ding ◽  
Susanne Schnabl ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Stromal Cells ◽  
Lymphocytic Leukemia ◽  
Marrow Stromal Cells ◽  
Parp Cleavage ◽  
Cell Contact ◽  
Human Mscs ◽  
Drug Induced ◽  
Wide Range ◽  
Induced Apoptosis

Abstract Marrow stromal cells (MSCs) provide important survival and drug resistance signals to chronic lymphocytic leukemia (CLL) cells, but current models to analyze CLL–MSC interactions are heterogeneous. Therefore, we tested different human and murine MSC lines and primary human MSCs for their ability to protect CLL cells from spontaneous and drug-induced apoptosis. Our results show that both human and murine MSCs are equally effective in protecting CLL cells from fludarabine-induced apoptosis. This protective effect was sustained over a wide range of CLL–MSC ratios (5:1 to 100:1), and the levels of protection were reproducible in 4 different laboratories. Human and murine MSCs also protected CLL cells from dexamethasone- and cyclophosphamide-induced apoptosis. This protection required cell–cell contact and was virtually absent when CLL cells were separated from the MSCs by micropore filters. Furthermore, MSCs maintained Mcl-1 and protected CLL cells from spontaneous and fludarabine-induced Mcl-1 and PARP cleavage. Collectively, these studies define common denominators for CLL cocultures with MSCs. They also provide a reliable, validated tool for future investigations into the mechanism of MSC–CLL cross talk and for drug testing in a more relevant fashion than the commonly used suspension cultures.

ONC201 Overcomes Chemotherapy Resistance By Upregulation of Bim in Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4476-4476 ◽  
Author(s):  
Yong-sheng Tu ◽  
Jin He ◽  
Huan Liu ◽  
Richard Eric Davis ◽  
Robert Z. Orlowski ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Cell Viability ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Parp Cleavage ◽  
Wild Type ◽  
Myeloma Cells ◽  
Induced Apoptosis ◽  
Tumor Types

Abstract In multiple myeloma, disease relapse and drug resistance occurs in the majority of myeloma patients after standard treatment despite recent improvements offered by new therapies. Therefore, there is an urgent need for new drugs that can overcome drug resistance and prolong patient survival after failure of standard therapies. ONC201, the founding member of a novel class of anti-tumor agents called impridones, has selective preclinical efficacy against a variety of tumor types. It is currently in phase I and phase II clinical trials for patients with advanced solid tumors and hematological malignancies. Given the pronounced sensitivity of B-cell lymphomas to ONC201, we assessed the efficacy of ONC201 in preclinical models of multiple myeloma. We treated human myeloma cell lines and primary myeloma cells isolated from bone marrow aspirates of myeloma patients with ONC201 for 72 hours. CellTiter-Glo Luminescent and annexin-V binding assays for assessing myeloma cell viability and apoptosis were performed, along with immunoblotting for cleavage of caspases, phosphorylation of signaling kinases, and expression of pro- or anti-apoptotic proteins. ONC201 treatment decreased myeloma cell viability, with IC50 values that were 1 μM to 1.5 μM, even in high risk myeloma cell line RPMI8226. The status of TP53 did not appear to affect the efficacy of ONC201, as MM.1S or NCI-H929 cells with wild-type TP53 and OPM-2 or RPMI8226 with mutated TP53 had a similar sensitivity towards ONC201. These results agree with prior reports in other tumor types that have demonstrated that the efficacy of ONC201 is independent of TP53. Western blot analysis showed increased apoptosis, cleavage of caspase-9, caspase-3, and PARP. We also found that ONC201 induced expression of the pro-apoptotic protein Bim in myeloma cells, which can occur downstream of ERK inactivation. Knockdown of Bim expression in myeloma cells by shRNAs abrogated ONC201-induced apoptosis. Phosphorylation of Bim at Ser69 by Erk1/2 has been shown to promote proteasomal degradation of Bim. In accordance with this mechanism, we observed that ONC201 treatment reduced levels of phosphorylated Erk1/2, an indicator of Erk1/2 kinase activity, and Bim pSer69. In addition, ONC201 induced apoptosis in dexamethasome-, bortezomib-, and carfilzomib-resistant myeloma cell lines with the same efficacy as in wild-type cells. As a rational strategy to increase the efficacy of ONC201 by enhancing its inhibition of proteasome-mediated Bim degradation, we tested combinations of ONC201 with proteasome inhibitors bortezomib or carfilzomib. These combinations were synergistic in reducing cell viability and enhancing Bim expression and PARP cleavage in myeloma cells. Overall, these findings demonstrate that ONC201 inhibits the Erk1/2 signaling pathway and induces Bim expression to induce apoptosis in multiple myeloma regardless of resistance to standard-of-care therapies. Our studies suggest that ONC201 should be evaluated clinically in relapsed/refractory multiple myeloma. Disclosures Allen: Oncoceutics: Employment, Equity Ownership.

Hybrid Compounds & Oxidative Stress Induced Apoptosis in Cancer Therapy

2020 ◽  
Vol 27 (13) ◽  
pp. 2118-2132 ◽  
Author(s):  
Aysegul Hanikoglu ◽  
Hakan Ozben ◽  
Ferhat Hanikoglu ◽  
Tomris Ozben
Keyword(s):  
Oxidative Stress ◽  
Drug Resistance ◽  
Side Effects ◽  
Cancer Therapy ◽  
Tumor Cells ◽  
Anticancer Drugs ◽  
Chemotherapeutic Agents ◽  
Oxidation Reactions ◽  
Hybrid Compounds ◽  
Induced Apoptosis

: Elevated Reactive Oxygen Species (ROS) generated by the conventional cancer therapies and the endogenous production of ROS have been observed in various types of cancers. In contrast to the harmful effects of oxidative stress in different pathologies other than cancer, ROS can speed anti-tumorigenic signaling and cause apoptosis of tumor cells via oxidative stress as demonstrated in several studies. The primary actions of antioxidants in cells are to provide a redox balance between reduction-oxidation reactions. Antioxidants in tumor cells can scavenge excess ROS, causing resistance to ROS induced apoptosis. Various chemotherapeutic drugs, in their clinical use, have evoked drug resistance and serious side effects. Consequently, drugs having single-targets are not able to provide an effective cancer therapy. Recently, developed hybrid anticancer drugs promise great therapeutic advantages due to their capacity to overcome the limitations encountered with conventional chemotherapeutic agents. Hybrid compounds have advantages in comparison to the single cancer drugs which have usually low solubility, adverse side effects, and drug resistance. This review addresses two important treatments strategies in cancer therapy: oxidative stress induced apoptosis and hybrid anticancer drugs.

Small Molecular Gemcitabine Prodrugs for Cancer Therapy

2020 ◽  
Vol 27 (33) ◽  
pp. 5562-5582 ◽  
Author(s):  
He Miao ◽  
Xuehong Chen ◽  
Yepeng Luan
Keyword(s):  
Drug Resistance ◽  
Side Effects ◽  
Anticancer Drug ◽  
Drug Stability ◽  
Effective Means ◽  
Active Form ◽  
Deoxycytidine Kinase ◽  
High Efficacy ◽  
Pyrimidine Nucleoside ◽  
Design And Synthesis

Gemcitabine as a pyrimidine nucleoside analog anticancer drug has high efficacy for a broad spectrum of solid tumors. Gemcitabine is activated within tumor cells by sequential phosphorylation carried out by deoxycytidine kinase to mono-, di-, and triphosphate nucleotides with the last one as the active form. But the instability, drug resistance and toxicity severely limited its utilization in clinics. In the field of medicinal chemistry, prodrugs have proven to be a very effective means for elevating drug stability and decrease undesirable side effects including the nucleoside anticancer drug such as gemcitabine. Many works have been accomplished in design and synthesis of gemcitabine prodrugs, majority of which were summarized in this review.

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