CDK Inhibitors Upregulate BH3-Only Proteins to Sensitize Human Myeloma Cells to BH3 Mimetic Therapies

Chen, Shuang; Dai, Yun; Pei, Xin Yan; Myers, Jennifer L.; Wang, Li; Kramer, Lora B.; Garnett, Mandy; Schwartz, Daniella M.; Su, Florence; Simmons, Gary; Richey, Justin D.; Larsen, Dustin G.; Dent, Paul; Orłowski, Robert Z.; Grant, Steven

doi:10.1158/0008-5472.can-12-1118

Cited by 53 publications

(63 citation statements)

References 49 publications

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“…Human multiple myeloma U266 and RPMI8226 cells were obtained from the ATCC and maintained as described previously (5), and both cell lines were authenticated (Basic STR Profiling Service, ATCC 135-X) by the ATCC before this study was completed. Bortezomibresistant cells (PS-R) were generated by continuously culturing U266 cells in increasing concentrations of bortezomib (beginning at 0.5 nM and increasing in stepwise increments of 0.2 nM) up to 20 nM and were maintained in medium containing 15 nM bortezomib.…”

Section: Cells and Reagentsmentioning

confidence: 99%

“…The extent of apoptosis was evaluated by flow cytometry utilizing annexin V-fluorescein isothiocyanate (FITC)-propidium iodide (PI) (5). Briefly, 1 ϫ 10 6 cells were stained with annexin V-FITC (BD PharMingen, San Diego, CA) and 5 g/ml PI (Sigma) in 1ϫ binding buffer for 15 min at room temperature in the dark.…”

Section: Cells and Reagentsmentioning

confidence: 99%

“…Total protein was quantified by using Coomassie protein assay reagent (Pierce, Rockford, IL). Equal amounts of protein (30 g) were subjected to immunoblotting according to procedures described previously (5). Where indicated, the blots were reprobed with antibodies against ␤-actin (Sigma) or ␣-tubulin (Oncogene, La Jolla, CA) to ensure equal loading and transfer of proteins.…”

Section: Autophagy Analysis Autophagy Analysis Was Performed As Follmentioning

confidence: 99%

“…SureSilencing short hairpin RNA (shRNA) plasmids (neomycin resistance) were purchased from SABioscience (Frederick, MD), which include shRNAs targeting SQSTM1 (GenBank accession number NM_003900; clone 4 ) and a scrambled sequence as a control (GGAATCTCATTCGATGC ATAC). U266 cells were stably transfected with these constructs by using an Amaxa Nucleofector device with Cell Line Specific Nucleofector kit C (Amaxa GmbH, Cologne, Germany) according to the manufacturer's instructions (5). The Lentiviral Particle Gene Silencers construct (sc-29390-V) encoding shRNA targeting human Lamp2 and control lentiviral particles were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and used to transduce RPMI8226 cells.…”

Section: Autophagy Analysis Autophagy Analysis Was Performed As Follmentioning

confidence: 99%

“…Autophagy and apoptosis share molecular regulatory mechanisms governed by Bcl-2 family proteins (3,4). Specifically, Bcl-2 and Bcl-x L prevent both apoptosis and autophagy by sequestering different BH3-only proteins (e.g., proapoptotic Bim and Bid [5] and proautophagic beclin-1 [6,7]). As a result, antagonism of Bcl-2/Bcl-x L function releases and activates these BH3-only proteins, leading to apoptosis and autophagy, respectively (8).…”

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confidence: 99%

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Targeting SQSTM1/p62 Induces Cargo Loading Failure and Converts Autophagy to Apoptosis via NBK/Bik

Chen

Zhou

Zhang

et al. 2014

Molecular and Cellular Biology

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eIn selective autophagy, the adaptor protein SQSTM1/p62 plays a critical role in recognizing/loading cargo (e.g., malfolded proteins) into autophagosomes for lysosomal degradation. Here we report that whereas SQSTM1/p62 levels fluctuated in a timedependent manner during autophagy, inhibition or knockdown of Cdk9/cyclin T1 transcriptionally downregulated SQSTM1/ p62 but did not affect autophagic flux. These interventions, or short hairpin RNA (shRNA) directly targeting SQSTM1/p62, resulted in cargo loading failure and inefficient autophagy, phenomena recently described for Huntington's disease neurons. These events led to the accumulation of the BH3-only protein NBK/Bik on endoplasmic reticulum (ER) membranes, most likely by blocking loading and autophagic degradation of NBK/Bik, culminating in apoptosis. Whereas NBK/Bik upregulation was further enhanced by disruption of distal autophagic events (e.g., autophagosome maturation) by chloroquine (CQ) or Lamp2 shRNA, it was substantially diminished by inhibition of autophagy initiation (e.g., genetically by shRNA targeting Ulk1, beclin-1, or Atg5 or pharmacologically by 3-methyladenine [3-MA] or spautin-1), arguing that NBK/Bik accumulation stems from inefficient autophagy. Finally, NBK/Bik knockdown markedly attenuated apoptosis in vitro and in vivo. Together, these findings identify novel cross talk between autophagy and apoptosis, wherein targeting SQSTM1/p62 converts cytoprotective autophagy to an inefficient form due to cargo loading failure, leading to NBK/Bik accumulation, which triggers apoptosis.A utophagy is an evolutionarily conserved process by which damaged organelles and unneeded proteins are degraded by lysosomes to maintain intracellular homeostasis and to recycle cellular nutrients. While autophagy can promote cell death (1), in most cases, it is cytoprotective and contributes to drug resistance (2). In response to chemotherapeutic agents, apoptosis (type I) and autophagy (type II) represent two major forms of programmed cell death (3). Autophagy and apoptosis share molecular regulatory mechanisms governed by Bcl-2 family proteins (3, 4). Specifically, Bcl-2 and Bcl-x L prevent both apoptosis and autophagy by sequestering different BH3-only proteins (e.g., proapoptotic Bim and Bid [5] and proautophagic beclin-1 [6,7]). As a result, antagonism of Bcl-2/Bcl-x L function releases and activates these BH3-only proteins, leading to apoptosis and autophagy, respectively (8). While apoptosis represents a well-established mechanism of action of conventional and targeted anticancer agents (3), autophagy may play both positive and negative roles in tumorigenesis and cancer treatment (9, 10). Consequently, whether autophagy should be inhibited or activated remains the subject of debate. Accordingly, both autophagy inhibitors and inducers are currently undergoing clinical evaluation (11).Protection of cells from injury by harmful macromolecules or damaged organelles through autophagy as a quality control (QC) mechanism (11, 12) involves the sequestration and tran...

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Section: Cells and Reagentsmentioning

confidence: 99%

Section: Cells and Reagentsmentioning

confidence: 99%

Section: Autophagy Analysis Autophagy Analysis Was Performed As Follmentioning

confidence: 99%

Section: Autophagy Analysis Autophagy Analysis Was Performed As Follmentioning

confidence: 99%

mentioning

confidence: 99%

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Targeting SQSTM1/p62 Induces Cargo Loading Failure and Converts Autophagy to Apoptosis via NBK/Bik

Chen

Zhou

Zhang

et al. 2014

Molecular and Cellular Biology

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Mitochondrial dysfunction RAD51, and Ku80 proteolysis promote apoptotic effects of Dinaciclib in Bcl‐xL silenced cells

Premkumar

Jane

Thambireddy

et al. 2017

Molecular Carcinogenesis

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In the present study, we investigated the effect of CDK inhibitors (ribociclib, palbociclib, seliciclib, AZD5438, and dinaciclib) on malignant human glioma cells for cell viability, apoptosis, oxidative stress, and mitochondrial function using various assays. None of the CDK inhibitors induced cell death at a clinically relevant concentration. However, low nanomolar concentrations of dinaciclib showed higher cytotoxic activity against Bcl-xL silenced cells in a time- and concentration-dependent manner. This effect was not seen with other CDK inhibitors. The apoptosis-inducing capability of dinaciclib in Bcl-xL silenced cells was evidenced by cell shrinkage, mitochondrial dysfunction, DNA damage, and increased phosphatidylserine externalization. Dinaciclib was found to disrupt mitochondrial membrane potential, resulting in the release of cytochrome c, AIF, and smac/DIABLO into the cytoplasm. This was accompanied by the downregulation of cyclin-D1, D3, and total Rb. Dinaciclib caused cell cycle arrest in a time- and concentration-dependent manner and with accumulation of cells in the sub-G1 phase. Our results also revealed that dinaciclib, but not ribociclib or palbociclib or seliciclib or AZD5438 induced intrinsic apoptosis via upregulation of the levels of pro-apoptotic proteins (Bax and Bak), resulting in the activation of caspases and cleavage of PARP. We also found an additional mechanism for the dinaciclib-induced augmentation of apoptosis due to abrogation RAD51-cyclin D1 interaction, specifically proteolysis of the DNA repair proteins RAD51 and Ku80. Our results suggest that successfully interfering with Bcl-xL function may restore sensitivity to dinaciclib and could hold the promise for an effective combination therapeutic strategy.

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Leading Small Molecule Inhibitors of Anti-Apoptotic Bcl-2 Family Members

Yazbeck

Johnson

2012

Cell Death Signaling in Cancer Biology and Treatment

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CDK Inhibitors Upregulate BH3-Only Proteins to Sensitize Human Myeloma Cells to BH3 Mimetic Therapies

Cited by 53 publications

References 49 publications

Targeting SQSTM1/p62 Induces Cargo Loading Failure and Converts Autophagy to Apoptosis via NBK/Bik

Targeting SQSTM1/p62 Induces Cargo Loading Failure and Converts Autophagy to Apoptosis via NBK/Bik

Mitochondrial dysfunction RAD51, and Ku80 proteolysis promote apoptotic effects of Dinaciclib in Bcl‐xL silenced cells

Leading Small Molecule Inhibitors of Anti-Apoptotic Bcl-2 Family Members

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