Phenylbutyrate interferes with the Fanconi anemia and BRCA pathway and sensitizes head and neck cancer cells to cisplatin

<p>Abstract</p> <p>Background</p> <p>Cisplatin has been widely used to treat head and neck cancer. One of the clinical limitations with this treatment, however, is that tumors that are initially responsive to cisplatin later acquire resistance. We have recently shown that a subset of head and neck cancer cell lines has a defective Fanconi anemia DNA damage response pathway and this defect correlates to cisplatin sensitivity. We have also shown that the histone deacetylase inhibitor phenylbutyrate sensitize human cells to cisplatin. In this study we explored whether phenylbutyrate may sensitize head and neck cancer cells by interfering with the Fanconi anemia pathway.</p> <p>Results</p> <p>We found that the phenylbutyrate sensitizes head and neck cancer cell lines to cisplatin. This sensitization by phenylbutyrate correlated to a significant decrease in the formation of cisplatin-induced FANCD2 nuclear foci, which is a functional read out of the Fanconi anemia and BRCA (FA/BRCA) pathway. This abrogation of the FA/BRCA pathway by phenylbutyrate was not due to loss of FANCD2 monoubiquitylation but rather correlated to a phenylbutyrate-mediated reduction in the expression of the BRCA1 protein. Furthermore, we found that cancer cells defective in the FA pathway were also sensitized to cisplatin by phenylbutyrate suggesting that phenylbutyrate targets additional pathways.</p> <p>Conclusion</p> <p>The results from this study suggest that phenylbutyrate may have therapeutic utility as a cisplatin sensitizer in head and neck cancer by inhibiting the FA/BRCA pathway through the down regulation of BRCA1 as well as by an FA/BRCA-independent mechanism.</p

Hsp90-binding immunophilins link p53 to dynein during p53 transport to the nucleus

The tumor suppressor protein p53 is known to be transported to the nucleus along microtubular tracks by cytoplasmic dynein. However, the connection between p53 and the dynein motor protein complex has not been established. Here, we show that hsp90.binding immunophilins link p53.hsp90 complexes to dynein and that prevention of that linkage in vivo inhibits the nuclear movement of p53. First, we show that p53.hsp90 heterocomplexes from DLD-1 human colon cancer cells contain an immunophilin (FKBP52, CyP-40, or PP5) as well as dynein. p53.hsp90.immunophilin.dynein complexes can be formed by incubating immunopurified p53 with rabbit reticulocyte lysate, and we show by peptide competition that the immunophilins link via their tetratricopeptide repeat domains to p53-bound hsp90 and by means of their PPIase domains to the dynein complex. The linkage of immunophilins to the dynein motor is indirect by means of the dynamitin component of the dynein-associated dynactin complex, and we show that purified FKBP52 binds directly by means of its PPIase domain to purified dynamitin. By using a temperature-sensitive mutant of p53 where cytoplasmic-nuclear movement occurs by shift to permissive temperature, we show that p53 movement is impeded when p53 binding to hsp90 is inhibited by the hsp90 inhibitor radicicol. Also, nuclear movement of p53 is inhibited when immunophilin binding to dynein is competed for by expression of a PPIase domain fragment in the same manner as when dynein linkage to cargo is dissociated by expression of dynamitin. This is the first demonstration of the linkage between an hsp90-chaperoned transcription factor and the system for its retrograde movement to the nucleus both in vitro and in vivo.Fil: Galigniana, Mario Daniel. University of Michigan; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Harrell, Jennifer M.. University of Michigan; Estados UnidosFil: O´Hagen, Heather M.. University of Michigan; Estados UnidosFil: Ljungman, Mats. University of Michigan; Estados UnidosFil: Pratt, William B.. University of Michigan; Estados Unido

Guadecitabine (SGI‐110) priming sensitizes hepatocellular carcinoma cells to oxaliplatin

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/135710/1/mol22015991799.pd

Membrane-elution analysis of content of cyclins A, B1, and E during the unperturbed mammalian cell cycle

<p>Abstract</p> <p>Background</p> <p>Problems with whole-culture synchronization methods for the study of the cell cycle have led to the need for an analysis of protein content during the cell cycle of cells that have not been starved or inhibited. The membrane-elution method is a method that allows the study of the cell cycle by producing a culture of unperturbed, synchronized cells.</p> <p>Results</p> <p>The Helmstetter membrane-elution method for the continuous production of newborn, unperturbed, mammalian cells has been enhanced so that the collection of cells of different cell cycle ages is automated, reproducible, and relatively inexpensive. We have applied the automated membrane-elution method to the analysis of cyclin content during the cell cycle. Cyclin E protein was invariant during the cell cycle. Cyclins B1 and A accumulated continuously during the cell cycle and were degraded at mitosis. Newborn cells had ~0.5% of the cyclin B1 content of dividing cells.</p> <p>Conclusion</p> <p>The expression patterns of cyclins A, B1, and E can be explained by constant mRNA levels during the cell cycle. Previously reported phase specific variations of the cyclins are not strictly necessary for cell-cycle progression. Cells produced by membrane-elution are available to other laboratories for analysis of the cell cycle.</p

Discovery and Mechanistic Elucidation of a Class of Protein Disulfide Isomerase Inhibitors for the Treatment of Glioblastoma

Protein disulfide isomerase (PDI) is overexpressed in glioblastoma, the most aggressive form of brain cancer, and folds nascent proteins responsible for the progression and spread of the disease. Herein we describe a novel nanomolar PDI inhibitor, pyrimidotriazinedione 35G8, that is toxic in a panel of human glioblastoma cell lines. We performed a medium‐throughput 20 000‐compound screen of a diverse subset of 1 000 000 compounds to identify cytotoxic small molecules. Cytotoxic compounds were screened for PDI inhibition, and, from the screen, 35G8 emerged as the most cytotoxic inhibitor of PDI. Bromouridine labeling and sequencing (Bru‐seq) of nascent RNA revealed that 35G8 induces nuclear factor‐like 2 (Nrf2) antioxidant response, endoplasmic reticulum (ER) stress response, and autophagy. Specifically, 35G8 upregulated heme oxygenase 1 and solute carrier family 7 member 11 (SLC7A11) transcription and protein expression and repressed PDI target genes such as thioredoxin‐interacting protein 1 (TXNIP) and early growth response 1 (EGR1). Interestingly, 35G8‐induced cell death did not proceed via apoptosis or necrosis, but by a mixture of autophagy and ferroptosis. Cumulatively, our data demonstrate a mechanism for a novel PDI inhibitor as a chemical probe to validate PDI as a target for brain cancer.Iron‐clad PDI inhibition: We describe a nanomolar, cytotoxic protein disulfide isomerase (PDI) inhibitor, 35G8, that is potent in a panel of human glioblastoma cell lines. Bromouridine‐labeling and sequencing of nascent RNA revealed that 35G8 induces Nrf2 antioxidant response, endoplasmic reticulum stress response, autophagy, and may induce cell death via ferroptosis.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141828/1/cmdc201700629.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/141828/2/cmdc201700629-sup-0001-misc_information.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/141828/3/cmdc201700629_am.pd

Risk factors for moderate-to-severe chronic graft-versus-host disease after allogeneic hematopoietic stem cell transplantation

AbstractAmong 810 consecutive hematopoietic stem cell transplantation (HSCT) patients, 679 survived more than 3 months and were evaluated for chronic GVHD. The aim of this study was to find predisposing factors increasing the risk of development of moderate-to-severe chronic GVHD. Many of the donors were HLA-identical siblings or related (n = 435), 185 were HLA-matched unrelated, and 59 were mismatched related or unrelated donors. Most of the patients had a hematological malignancy (n = 568), but 111 patients with a nonmalignant disease were included. Two hundred twenty-three patients (33%) developed mild, 41 (6%) moderate, and 15 (2.2%) severe chronic GVHD. The 5-year probability of development of moderate-to-severe chronic GVHD was 10%. We analyzed 30 potential risk factors for chronic GVHD. In the multivariate analysis, acute GVHD grades II to IV (relative hazard [RH], 2.30; 95% CI, 1.29- 4.10; P = .005), CML diagnosis (RH, 2.37; CI, 1.38-4.08; P = .002) and transplantation from an immunized female donor to a male recipient (RH, 2.16; CI, 1.14-4.11; P = .02) were independent risk factors for moderate-to-severe chronic GVHD. Recipient age also was significant (RH, 2.42; CI, 1.23-4.77; P = .01) if CML was not included in the analysis. In patients with no risk factors, the 5-year probability of development of moderate-to-severe chronic GVHD was 5%. In patients with 1 risk factor, the probability was 13%; 2 risk factors, 23%; and 3 risk factors, 45%. Among patients who developed chronic GVHD (n = 279), acute GVHD grades II to IV (RH, 2.18; CI, 1.23-3.86; P < .01) was the only predictive factor for moderate-to-severe chronic GVHD versus mild disease. Patients with previous acute GVHD grades II to IV may benefit from more aggressive initial treatment. This possibility would have to be examined in clinical trials.Biol Blood Marrow Transplant 2002;8(12):674-82

The p53-targeting human phosphatase hCdc14A interacts with the Cdk1/cyclin B complex and is differentially expressed in human cancers

BACKGROUND: The evolutionary conserved cyclin-dependent kinase phosphatase hCdc14A has been shown to play potential roles in the regulation of mitotic exit and in the centrosome duplication cycle. We have recently shown that hCdc14A also can interact with the tumor suppressor p53 both in vitro and in vivo and specifically dephosphorylates the ser315 site of p53 in vitro. In this study we developed antibodies against hCdc14A to investigate the expression and regulation of hCdc14A in human tissues and cancer cells. RESULTS: We show that hCdc14A is differentially expressed in human tissues and in 75 cancer cell lines examined. Treatments with the histone deacetylase inhibitor TSA, the demethylating agent 5-aza-2'-deoxycytodine or the proteasome inhibitor MG132 significantly induced expression of hCdc14A in cell lines expressing low or undetectable levels of hCdc14A. There was a strong bias for low expression of hCdc14A in cancer cell lines harboring wild-type p53, suggesting that high Cdc14A expression is not compatible with wild-type p53 expression. We present evidence for a role for hCdc14A in the dephosphorylation of the ser315 site of p53 in vivo and that hCdc14A forms a complex with Cdk1/cyclin B during interphase but not during mitosis. CONCLUSION: Our results that hCdc14A is differentially expressed in human cancer cells and that hCdc14A can interact with both p53 and the Cdk1/cyclin B complex may implicate that dysregulation of hCdc14A expression may play a role in carcinogenesis

Pifithrin-alpha inhibits p53 signaling after interaction of the tumor suppressor protein with hsp90 and its nuclear translocation

Pifithrin-alpha (PFTalpha) was originally thought to be a specific inhibitor of signaling by the tumor suppressor protein p53. However, the laboratory that discovered pifithrin recently reported that the compound also inhibits heat shock and glucocorticoid receptor (GR) signaling, and they suggested that PFTalpha targets a factor common to all three signal transduction pathways, such as the hsp90/hsp70-based chaperone machinery (Komarova, E. A., Neznanov, N., Komarov, P. G., Chernov, M. V., Wang, K., and Gudkov, A. V. (2003) J. Biol. Chem. 278, 15465-15468). Because it is important for the mechanistic study of this machinery to identify unique inhibitors of chaperone action, we have examined the effect of PFTalpha on transcriptional activation, the hsp90 heterocomplex assembly, and hsp90-dependent nuclear translocation for both p53 and the GR. At concentrations where PFTalpha blocks p53-mediated induction of p21/Waf-1 in human embryonic kidney cells, we observed no inhibition of GR-mediated induction of a chloramphenicol acetyl transferase reporter in LMCAT cells. PFTalpha did, however, cause a left shift in the dexamethasone dose response curve by increasing intracellular dexamethasone concentration, apparently by competing for dexamethasone efflux from the cell. The assembly of p53 or GR heterocomplexes with hsp90 and immunophilins was not affected by PFTalpha either in vivo or in vitro and did not affect the nuclear translocation of either transcription factor. Thus, we conclude that PFTalpha does not inhibit GR-mediated induction or the function of the chaperone machinery, and, as originally thought, it may specifically inhibit p53 signaling by acting at a stage after p53 translocation to the nucleus.Fil: Murphy, Patrick J.. University of Michigan; Estados UnidosFil: Galigniana, Mario Daniel. University of Michigan; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Morishima, Yoshihiro. University of Michigan; Estados UnidosFil: Harrell, Jennifer M.. University of Michigan; Estados UnidosFil: Kwok, Roland P.. University of Michigan; Estados UnidosFil: Ljungman, Mats. University of Michigan; Estados UnidosFil: Pratt, William B.. University of Michigan; Estados Unido

Mutant p53R270H drives altered metabolism and increased invasion in pancreatic ductal adenocarcinoma

Pancreatic cancer is characterized by nearly universal activating mutations in KRAS. Among other somatic mutations, TP53 is mutated in more than 75% of human pancreatic tumors. Genetically engineered mice have proven instrumental in studies of the contribution of individual genes to carcinogenesis. Oncogenic Kras mutations occur early during pancreatic carcinogenesis and are considered an initiating event. In contrast, mutations in p53 occur later during tumor progression. In our model, we recapitulated the order of mutations of the human disease, with p53 mutation following expression of oncogenic Kras. Further, using an inducible and reversible expression allele for mutant p53, we inactivated its expression at different stages of carcinogenesis. Notably, the function of mutant p53 changes at different stages of carcinogenesis. Our work establishes a requirement for mutant p53 for the formation and maintenance of pancreatic cancer precursor lesions. In tumors, mutant p53 becomes dispensable for growth. However, it maintains the altered metabolism that characterizes pancreatic cancer and mediates its malignant potential. Further, mutant p53 promotes epithelial-mesenchymal transition (EMT) and cancer cell invasion. This work generates new mouse models that mimic human pancreatic cancer and expands our understanding of the role of p53 mutation, common in the majority of human malignancies