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1.  Targeting ER stress–induced autophagy overcomes BRAF inhibitor resistance in melanoma 
The Journal of Clinical Investigation  2014;124(3):1406-1417.
Melanomas that result from mutations in the gene encoding BRAF often become resistant to BRAF inhibition (BRAFi), with multiple mechanisms contributing to resistance. While therapy-induced autophagy promotes resistance to a number of therapies, especially those that target PI3K/mTOR signaling, its role as an adaptive resistance mechanism to BRAFi is not well characterized. Using tumor biopsies from BRAFV600E melanoma patients treated either with BRAFi or with combined BRAF and MEK inhibition, we found that BRAFi-resistant tumors had increased levels of autophagy compared with baseline. Patients with higher levels of therapy-induced autophagy had drastically lower response rates to BRAFi and a shorter duration of progression-free survival. In BRAFV600E melanoma cell lines, BRAFi or BRAF/MEK inhibition induced cytoprotective autophagy, and autophagy inhibition enhanced BRAFi-induced cell death. Shortly after BRAF inhibitor treatment in melanoma cell lines, mutant BRAF bound the ER stress gatekeeper GRP78, which rapidly expanded the ER. Disassociation of GRP78 from the PKR-like ER-kinase (PERK) promoted a PERK-dependent ER stress response that subsequently activated cytoprotective autophagy. Combined BRAF and autophagy inhibition promoted tumor regression in BRAFi-resistant xenografts. These data identify a molecular pathway for drug resistance connecting BRAFi, the ER stress response, and autophagy and provide a rationale for combination approaches targeting this resistance pathway.
doi:10.1172/JCI70454
PMCID: PMC3934165  PMID: 24569374
2.  Off-target Lapatinib Activity Sensitizes Colon Cancer Cells through TRAIL Death Receptor Up-regulation 
Science translational medicine  2011;3(86):86ra50.
Lapatinib, a HER2/EGFR inhibitor, is a recently approved targeted therapy for metastatic breast cancer. As lapatinib enhances the efficacy of the antimetabolite capecitabine in breast cancer patients, we lapatinib also enhance the activity of anti-cancer agents in colorectal cancer. We found that lapatinib the pro-apoptotic effects of Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL) and TRAIL receptor antibodies mapatumumab and lexatumumab. Tumors from mice treated with lapatinibTRAIL exhibited more immunostaining for cleaved caspase-8, the extrinsic cell death pathway, tumors from mice treated with lapatinib or TRAIL alone. Furthermore, combination therapy suppressed tumor growth more effectively than treatment. apatinib up-the proapoptotic TRAIL death receptors DR4 and DR5, leading to more efficient induction of apoptosis in the presence of TRAIL receptor agonistsThis activity was independent of EGFR and HER2 off-target induction of DR5 by lapatinib activation of the JNK/c-Jun signaling axis. This activity of lapatinib on TRAIL death receptor expression and signaling may confer therapeutic benefit when increased doses of lapatinib are used in combination with TRAIL-receptor-activating agents.
doi:10.1126/scitranslmed.3001384
PMCID: PMC3769950  PMID: 21653830
3.  Overcoming hypoxia-induced apoptotic resistance through combinatorial inhibition of GSK-3β and CDK1 
Cancer research  2011;71(15):5265-5275.
Tumor hypoxia is an inherent impediment to cancer treatment that is both clinically significant and problematic. In this study, we performed a cell-based screen to identify small molecules that could reverse the apoptotic resistance of hypoxic cancer cells. Among the compounds we identified were a structurally-related group that sensitized hypoxic cancer cells to apoptosis by inhibiting the kinases GSK-3β and CDK1. Combinatorial inhibition of these proteins in hypoxic cancer cells and tumors increased levels of c-Myc and decreased expression of c-IAP2 and the central hypoxia response regulator Hif-1α. In mice, these compounds augmented the hypoxic tumor cell death induced by cytotoxic chemotherapy, blocking angiogenesis and tumor growth. Taken together, our findings suggest that combinatorial inhibition of GSK-3β and CDK1 augment the apoptotic sensitivity of hypoxic tumors, and they offer preclinical validation of a novel and readily translatable strategy to improve cancer therapy.
doi:10.1158/0008-5472.CAN-11-1383
PMCID: PMC3667402  PMID: 21646472
GSK-3β; CDK1; c-Myc; Hif-1α; c-IAP2; hypoxia; apoptosis; drug screen; drug resistance
4.  ER stress–mediated autophagy promotes Myc-dependent transformation and tumor growth 
The Journal of Clinical Investigation  2012;122(12):4621-4634.
The proto-oncogene c-Myc paradoxically activates both proliferation and apoptosis. In the pathogenic state, c-Myc–induced apoptosis is bypassed via a critical, yet poorly understood escape mechanism that promotes cellular transformation and tumorigenesis. The accumulation of unfolded proteins in the ER initiates a cellular stress program termed the unfolded protein response (UPR) to support cell survival. Analysis of spontaneous mouse and human lymphomas demonstrated significantly higher levels of UPR activation compared with normal tissues. Using multiple genetic models, we demonstrated that c-Myc and N-Myc activated the PERK/eIF2α/ATF4 arm of the UPR, leading to increased cell survival via the induction of cytoprotective autophagy. Inhibition of PERK significantly reduced Myc-induced autophagy, colony formation, and tumor formation. Moreover, pharmacologic or genetic inhibition of autophagy resulted in increased Myc-dependent apoptosis. Mechanistically, we demonstrated an important link between Myc-dependent increases in protein synthesis and UPR activation. Specifically, by employing a mouse minute (L24+/–) mutant, which resulted in wild-type levels of protein synthesis and attenuation of Myc-induced lymphomagenesis, we showed that Myc-induced UPR activation was reversed. Our findings establish a role for UPR as an enhancer of c-Myc–induced transformation and suggest that UPR inhibition may be particularly effective against malignancies characterized by c-Myc overexpression.
doi:10.1172/JCI62973
PMCID: PMC3533536  PMID: 23143306
5.  Human colon cancer stem cells are enriched by insulin-like growth factor-1 and are sensitive to figitumumab 
Cell Cycle  2011;10(14):2331-2338.
Cancer stem cells (CSCs) are recognized as contributors to cancer progression and therapeutic resistance in liquid and solid malignancies. We analyzed a panel of human colon cancer cell lines for CSC populations by side population and aldehyde dehydrogenase activity. IGF-1 enriches these putative colon CSC populations in a β-catenin-dependent manner. Chemical inhibition of Akt depletes SP cells, and conversely, the overexpression of a constitutively active mutant version of Akt is sufficient to enrich CSC populations. CP-751,871, a fully human antibody with specificity to the IGF-1 receptor, is currently being tested in clinical trials for a variety of solid tumors. CP-751,871 reduces CSC populations in colon cancer cell lines in vitro and reduces tumor growth in vivo. We have identified a novel role for IGF-1 in the enrichment of chemoresistant CSC populations. Our results suggest that CP-751,871 has preferential activity against putative CSC populations and, therefore, may complement current standard chemotherapeutic regimens that target cycling cells.
doi:10.4161/cc.10.14.16418
PMCID: PMC3322474  PMID: 21720213
IGF-1; cancer stem cell; colon cancer; figitumumab
6.  Visualization and enrichment of live putative cancer stem cell populations following p53 inactivation or Bax deletion using non-toxic fluorescent dyes 
Cancer biology & therapy  2009;8(22):2194-2205.
Putative cancer stem cell (CSC) populations efflux dyes such as Hoechst 33342 giving rise to side populations (SP) that can be analyzed or isolated by flow cytometry. However, Hoechst 33342 is highly toxic, more so to non-SP cells, and thus presents difficulties in interpreting in vivo studies where non-SP cells appear less tumorigenic than SP cells in immunodeficient mice. We searched for non-toxic dyes to circumvent this problem as well as to image these putative CSCs. We found that the fluorescent dye calcein, a product of intracellular Calcein AM cleavage, is effluxed by a small subpopulation, calcein low population (CloP). This population overlaps with SP and demonstrated long term cell viability, lack of cell stress and proliferation in several cancer cell lines when stained whereas Hoechst 33342 staining caused substantial apoptosis and ablated proliferation. We also found that the effluxed dye D-luciferin exhibits strong UV-fluorescence that can be imaged at cellular resolution and spatially overlaps with Calcein AM. In order to evaluate the hypothesis that p53 loss promotes enrichment of putative CSC populations we used Calcein AM, D-luciferin and Mitotracker Red FM as a counterstain to visualize dye-effluxing cells. Using fluorescence microscopy and flow cytometry we observed increased dye-effluxing populations in DLD-1 colon tumor cells with mutant p53 versus wild-type (WT) p53-expressing HCT116 cells. Deletion of the wild-type p53 or pro-apoptotic Bax genes induced the putative CSC populations in the HCT116 background to significant levels. Restoration of WT p53 in HCT116 p53−/− cells by an adenovirus vector eliminated the putative CSC populations whereas a control adenovirus vector, Ad-LacZ, maintained the putative CSC population. Our results suggest it is possible to image and quantitatively analyze putative CSC populations within the tumor microenvironment and that loss of pro-apoptotic and tumor suppressing genes such as Bax or p53 enrich such tumor-prone populations.
PMCID: PMC2975270  PMID: 19923899
cancer stem cells; p53; Hoechst 33342; Calcein AM; Bax; colon cancer; microscopy; flow cytometry; side population; calcein low population
7.  The p53 target Plk2 interacts with TSC proteins impacting mTOR signaling, tumor growth and chemosensitivity under hypoxic conditions 
Cell cycle (Georgetown, Tex.)  2009;8(24):4168-4175.
Tuberous sclerosis complex 1 (TSC1) inhibits mammalian target of rapamycin (mTOR), a central promotor of cell growth and proliferation. The protein product of the TSC1 gene, hamartin (referred to as TSC1) is known to interact with Polo-like kinase 1 (Plk1) in a cell cycle regulated, phosphorylation-dependent manner. We hypothesized that the p53 target gene, Plk2, is a tumor suppressor, mediating its tumor suppressor function through interactions with TSC1 that facilitate TSC1/2 restraint of mTOR under hypoxic stress. We found that human lung tumor cells deficient in Plk2 grew larger than control tumors, and that Plk2 interacts with endogenous TSC1 protein. Additionally, C-terminal Plk2-GST fusion protein bound both TSC1 and TSC2 proteins. TSC1 levels were elevated in response to Adriamycin and cells transiently overexpressing Plk2 demonstrated decreased phosphorylation of the downstream target of mTOR, ribosomal protein p70S6 kinase during hypoxia. Plk2 levels were inversely correlated with cytoplasmic p70S6K phosphorylation. Plk2 levels did not increase in response to DNA damage (Adriamycin, CPT-11) when HCT 116 and H460 cells were exposed to hypoxia. TSC1-deficient mouse embryonic fibroblasts with TSC1 added back demonstrated decreased S6K phosphorylation, which was further decreased when Plk2 was transiently overexpressed. Interestingly, under normoxia, Plk2 deficient tumor cells demonstrated increased apoptosis in response to various chemotherapeutic agents including CPT-11 but increased resistance to apoptotic death after CPT-11 treatment under hypoxia, and tumor xenografts comprised of these Plk2-deficient cells were resistant to CPT-11. Our results point to a novel Plk2-TSC1 interaction with effects on mTOR signaling during hypoxia, and tumor growth that may enable targeting Plk2 signaling in cancer therapy.
PMCID: PMC2975271  PMID: 20054236
polo-like kinase (Plk2); p53; tuberous sclerosis complex 1 (TSC1); mammalian target of rapamycin (mTOR); hypoxia; p70S6 kinase (p70S6K); tuberous sclerosis complex 2 (TSC2)
8.  OSU-03012 Stimulates PKR-Like Endoplasmic Reticulum-Dependent Increases in 70-kDa Heat Shock Protein Expression, Attenuating Its Lethal Actions in Transformed Cells 
Molecular pharmacology  2008;73(4):1168-1184.
We have further defined mechanism(s) by which 2-amino-N-{4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-phenyl}acetamide [OSU-03012 (OSU)], a derivative of the cyclooxygenase-2 (COX2) inhibitor celecoxib but lacking COX2 inhibitory activity, kills transformed cells. In cells lacking expression of protein kinase R-like endoplasmic reticulum kinase (PERK-/-), the lethality of OSU was attenuated. OSU enhanced the expression of Beclin 1 and ATG5 and cleavage of pro-caspase 4 in a PERK-dependent fashion and promoted the Beclin 1- and ATG5-dependent formation of vacuoles containing LC3, followed by a subsequent caspase 4-dependent cleavage of cathepsin B and a cathepsin B-dependent formation of low pH intracellular vesicles; cathepsin B was activated and released into the cytosol and genetic suppression of caspase 4, cathepsin B, or apoptosis-inducing factor function significantly suppressed cell killing. In parallel, OSU caused PERK-dependent increases in 70-kDa heat shock protein (HSP70) expression and decreases in 90-kDa heat shock protein (HSP90) and Grp78/BiP expression. Changes in HSP70 expression were post-transcriptional. Knockdown or small-molecule inhibition of HSP70 expression enhanced OSU toxicity, and overexpression of HSP70 suppressed OSU-induced low pH vesicle formation and lethality. Our data demonstrate that OSU-03012 causes cell killing that is dependent on PERK-induced activation of multiple toxic proteases. OSU-03012 also increased expression of HSP70 in a PERK-dependent fashion, providing support for the contention that OSU-03012-induced PERK signaling promotes both cell survival and cell death processes.
doi:10.1124/mol.107.042697
PMCID: PMC2674576  PMID: 18182481

Results 1-8 (8)