Gu Xiao

DIFFERENTIAL ACTIVATION OF P53 BY THE VARIOUS ADDUCTS OF MITOMYCIN C

Mitomycin C (MC) is a cytotoxic chemotherapeutic agent that causes DNA damage in the form of DNA cross-links as well as a variety of DNA monoadducts and is known to induce p53. The various DNA adducts formed upon treatment of mouse mammary tumor cells with MC as well as 10-decarbamoyl MC (DMC) and 2,7-diaminomitosene (2,7-DAM), the major MC metabolite, have been elucidated. The cytotoxicity of DMC parallels closely that of MC in a number of rodent cell lines tested, whereas 2,7-DAM is relatively noncytotoxic. In this study, we investigate the ability of MC, DMC, and 2,7-DAM to activate p53 at equidose concentrations by treating tissue culture cell lines with the three mitomycins. Whereas MC and DMC induced p53 protein levels and increased the levels of p21 and Gadd45 mRNA, 2,7-DAM did not. Furthermore, MC and DMC, but not 2,7-DAM, were able to induce apoptosis efficiently in ML-1 cells. Therefore the 2,7-DAM monoadducts were unable to activate the p53 pathway. Interestingly, DMC was able to initiate apoptosis via a p53-independent pathway whereas MC was not. This is the first finding that adducts of a multiadduct type DNA-damaging agent are differentially recognized by DNA damage sensor pathways.

Proteome-wide analysis of mutant p53 targets in breast cancer identifies new levels of gain-of-function that influence PARP, PCNA, and MCM4

Significance Mutant p53 (mtp53) is a driver oncogene of breast cancer. Here, for the first time, to our knowledge, using an inducible endogenous knockdown system, we explore the mtp53 driven proteome. We report this key data set that highlights mtp53-driven proteome diversity at the level of protein localization, as well as changes in protein levels without corresponding changes in transcription. We validated two protein pathways that include increased chromatin association of poly(ADP ribose) polymerase 1, and the increase of nuclear replication proteins minichromosome maintenance 4 and proliferating cell nuclear antigen. The addition of mtp53 proteomic targets to the previously identified transcriptional targets suggests that effective treatment of mtp53-driven breast cancers may be facilitated by new combination protocols blocking proteins of the metabolic pathways of cholesterol biosynthesis, DNA replication, and DNA repair. The gain-of-function mutant p53 (mtp53) transcriptome has been studied, but, to date, no detailed analysis of the mtp53-associated proteome has been described. We coupled cell fractionation with stable isotope labeling with amino acids in cell culture (SILAC) and inducible knockdown of endogenous mtp53 to determine the mtp53-driven proteome. Our fractionation data highlight the underappreciated biology that missense mtp53 proteins R273H, R280K, and L194F are tightly associated with chromatin. Using SILAC coupled to tandem MS, we identified that R273H mtp53 expression in MDA-MB-468 breast cancer cells up- and down-regulated multiple proteins and metabolic pathways. Here we provide the data set obtained from sequencing 73,154 peptide pairs that then corresponded to 3,010 proteins detected under reciprocal labeling conditions. Importantly, the high impact regulated targets included the previously identified transcriptionally regulated mevalonate pathway proteins but also identified two new levels of mtp53 protein regulation for nontranscriptional targets. Interestingly, mtp53 depletion profoundly influenced poly(ADP ribose) polymerase 1 (PARP1) localization, with increased cytoplasmic and decreased chromatin-associated protein. An enzymatic PARP shift occurred with high mtp53 expression, resulting in increased poly-ADP-ribosylated proteins in the nucleus. Mtp53 increased the level of proliferating cell nuclear antigen (PCNA) and minichromosome maintenance 4 (MCM4) proteins without changing the amount of pcna and mcm4 transcripts. Pathway enrichment analysis ranked the DNA replication pathway above the cholesterol biosynthesis pathway as a R273H mtp53 activated proteomic target. Knowledge of the proteome diversity driven by mtp53 suggests that DNA replication and repair pathways are major targets of mtp53 and highlights consideration of combination chemotherapeutic strategies targeting cholesterol biosynthesis and PARP inhibition.

p53 binds to a constitutively nucleosome free region of the mdm2 gene

The mdm2 oncogene is a p53 responsive gene which contains both a p53 independent and a p53 dependent promoter (P1 and P2 respectively). We have utilized ligation mediated PCR genomic footprinting in order to investigate the intra-nuclear binding of p53 to the mdm2 P2 promoter. The DNase I protection pattern in nuclei from murine cells lacking p53 has been compared to the protection pattern in cells containing a temperature sensitive p53-Val135. At 32°C p53-Val135 assumes a wild-type conformation while at 37°C this p53 is conformationally mutant. We observed clear wild-type p53 dependent protection of the putative p53 response elements (REs) as well as protection of the adjacent TATA box. Interestingly the protection pattern observed with purified wild-type p53 on naked DNA showed less nucleotide sequence protection than the protection observed to be p53 dependent in nuclei. Constitutive DNase I hypersensitivity at both the mdm2 P1 and P2 promoters was detected by indirect Southern blot analysis. DNase I hypersensitivity reflects altered chromatin conformations resulting, most likely, from the absence of nucleosomes. Taken together our findings suggest that the mdm2 P2 promoter is maintained in a nucleosome free state which is pre-primed for transcriptional activation by p53.

Decarbamoyl mitomycin C (DMC) activates p53-independent ataxia telangiectasia and rad3 related protein (ATR) chromatin eviction

Interstrand crosslinks induce DNA replication fork stalling that in turn activates the ATR-dependent checkpoint and DNA repair on nuclear chromatin. Mitomycin C (MC) and Decarbamoyl Mitomycin C (DMC) induce different types of DNA crosslinks with DMC being a more cytotoxic agent. We previously reported that the novel DMC induced β–interstrand DNA crosslinks induce a p53-independent form of cell death. The p53-independent DMC cytotoxicity associates with the activation, and subsequent depletion, of Chk1. In this study we further dissect the novel DMC signal transduction pathway and asked how it influences chromatin-associated proteins. We found that treatment with DMC, but not MC, stimulated the disassociation of ATR from chromatin and re-localization of ATR to the cytoplasm. The chromatin eviction of ATR was coupled with the formation of nuclear Rad51 foci and the phosphorylation of Chk1. Furthermore, DMC but not MC, activated expression of gadd45α mRNA. Importantly, knocking down p53 via shRNA did not inhibit the DMC-induced disassociation of ATR from chromatin or reduce the activation of transcription of gadd45α. Our results suggest that DMC induces a p53-independent disassociation of ATR from chromatin that facilitates Chk1 checkpoint activation and Rad51 chromatin recruitment. Our findings provide evidence that ATR chromatin eviction in breast cancer cells is an area of study that should be focused on for inducing p53-independent cell death.

Identification, validation, and targeting of the mutant p53-PARP-MCM chromatin axis in triple negative breast cancer

Over 80% of triple negative breast cancers express mutant p53. Mutant p53 often gains oncogenic function suggesting that triple negative breast cancers may be driven by p53 protein type. To determine the chromatin targets of this gain-of-function mutant p53 we used inducible knockdown of endogenous gain-of-function mtp53 in MDA-MB-468 cells in conjunction with stable isotope labeling with amino acids in cell culture and subcellular fractionation. We sequenced over 70,000 total peptides for each corresponding reciprocal data set and were able to identify 3010 unique cytoplasmic fraction proteins and 3403 unique chromatin fraction proteins. The present proteomics experiment corroborated our previous experiment-based results that poly ADP-ribose polymerase has a positive association with mutant p53 on the chromatin. Here, for the first time we report that the heterohexomeric minichromosome maintenance complex that participates in DNA replication initiation ranked as a high mutant p53-chromatin associated pathway. Enrichment analysis identified the minichromosome maintenance members 2–7. To validate this mutant p53- poly ADP-ribose polymerase-minichromosome maintenance functional axis, we experimentally depleted R273H mutant p53 and found a large reduction of the amount of minichromosome maintenance complex proteins on the chromatin. Furthermore a mutant p53-minichromosome maintenance 2 direct interaction was detected. Overexpressed mutant p53, but not wild type p53, showed a protein-protein interaction with minichromosome maintenance 2 and minichromosome maintenance 4. To target the mutant p53- poly ADP-ribose polymerase-minichromosome maintenance axis we treated cells with the poly ADP-ribose polymerase inhibitor talazoparib and the alkylating agent temozolomide and detected synergistic activation of apoptosis only in the presence of mutant p53. Furthermore when minichromosome maintenance 2–7 activity was inhibited the synergistic activation of apoptosis was blocked. This mutant p53- poly ADP-ribose polymerase -minichromosome maintenance axis may be useful for theranostics.Personalized medicine: Mutated tumors respond to therapyMutations in the p53 tumor suppressor gene could offer a predictive biomarker of response to certain drugs in triple-negative breast cancer. Jill Bargonetti from Hunter College in New York, USA, and colleagues showed that mutant p53, which is expressed in more than 80% of patients with triple-negative breast cancer, interacts with and regulates hundreds of proteins, including those found in a complex needed for DNA replication. Members of this complex, called the minichromosome maintenance protein complex, interact with mutant p53—but less with wild-type p53. Bargonetti’s team targeted this pathway in mutated breast cancer cells with the PARP inhibitor talazoparib and the chemotherapeutic agent temozolomide. They observed synergistic cell killing with the two drugs, but only when the minichromosome maintenance protein complex was working and when p53 was mutated. These findings point toward a new strategy for personalizing therapy.

Estrogen-activated MDM2 disrupts mammary tissue architecture through a p53-independent pathway

The Cancer Genome Atlas (TCGA) data indicate that high MDM2 expression correlates with all subtypes of breast cancer. Overexpression of MDM2 drives breast oncogenesis in the presence of wild-type or mutant p53 (mtp53). Importantly, estrogen-receptor positive (ER+) breast cancers overexpress MDM2 and estrogen mediates this expression. We previously demonstrated that this estrogen-MDM2 axis activates the proliferation of breast cancer cell lines T47D (mtp53 L194F) and MCF7 (wild-type p53) in a manner independent of increased degradation of wild-type p53 (ie, p53-independently). Herein we present data supporting the role of the estrogen-MDM2 axis in regulating cell proliferation and mammary tissue architecture of MCF7 and T47D cells in a p53-independent manner. Inducible shRNA mediated MDM2 knockdown inhibited colony formation in soft agar, decreased mass size and induced lumen formation in matrigel and also significantly reduced mitosis as seen by decreased phospho-histone H3 positive cells. The knockdown of MDM2 in both cell lines decreased Rb phosphorylation and the level of E2F1 protein. This signaling was through the estrogen receptor because fulvestrant (a selective estrogen receptor degrader) decreased MDM2 protein levels and decreased phosphorylation of Rb. Taken together these data indicate that in some ER+ breast cancers the estrogen-MDM2-Rb-E2F1 axis is a central hub for estrogen-mediated p53-independent signal transduction. This is the first indication that estrogen signaling utilizes the estrogen-MDM2 axis to provoke phosphorylation of Rb and increase E2F1 while promoting abnormal mammary architecture.

Abstract 2493: Identification of the mutant p53-PARP-MCM chromatin axis as a triple negative breast cancer replication stress target

Approximately 15% of all breast cancer is triple-negative and of these about 80% are found to have mutations in the gene for the tumor suppressor p53 (TP53). Many TP53 mutations encode gain-of-function oncogenic mutant p53 (GOF mtp53) protein. We used inducible knockdown of endogenous GOF mtp53 in MDA-MB-468 cells in conjunction with stable isotope labeling with amino acids in cell culture (SILAC) and subcellular fractionation. We sequenced over 70,000 total peptides for chromatin and cytoplasmic reciprocal data sets and were able to identify 3,010 unique cytoplasmic fraction proteins and 3,403 unique chromatin fraction proteins. We found that the heterohexomeric minichromosome maintenance (MCM) complex (MCM 2-7) along with PARP are high mtp53-chromatin associated pathways. When we depleted R273H mtp53 we found a large reduction of the amount of MCM complex and PARP proteins on the chromatin. Furthermore a direct mtp53-MCM2 interaction was detected. Overexpressed mtp53, but not wild type p53, showed a protein-protein interaction with MCM2 and MCM4. We treated cells with the PARP inhibitor talazoparib and the alkylating agent temozolomide and detected synergistic activation of apoptosis only in the presence of functional MCM2-7 and mtp53. The mtp53-PARP-MCM axis has potential use as a therapeutic and diagnostic target. Citation Format: Wei-Gang Qiu, Alla Polotskaia, Gu Xiao, Lia Di, Yuhan Zhao, Wenwei Hu, John Philip, Ronald Hendrickson, Jill Bargonetti. Identification of the mutant p53-PARP-MCM chromatin axis as a triple negative breast cancer replication stress target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2493. doi:10.1158/1538-7445.AM2017-2493

Abstract 3684: Proteome-wide triple negative breast cancer mutant p53 association index identifies chromatin unwinding for precision therapeutics

Over 80% of TNBCs express mutant p53 (mtp53) proteins. We coupled cell fractionation with stable isotope labeling with amino acids in cell culture (SILAC) and inducible knockdown of endogenous mtp53 to determine the mtp53 driven proteome in the cytoplasm and chromatin of triple negative breast cancer (TNBC) cells. Using SILAC coupled to tandem mass spectrometry (LC-MS/MS) we identified that R273H mtp53 expression in MDA-MB-468 breast cancer cells both up and down-regulated multiple proteins and metabolic pathways. We sequenced 73,154 peptide pairs that corresponded to 3010 proteins detected under reciprocal labeling conditions in the cytoplasm and 48,825 peptide pairs that corresponded to 5195 proteins in the chromatin fraction. Pathway enrichment analysis ranked the DNA unwinding pathway as the highest chromatin associated pathway regulated by mtp53. Moreover, to summarize and quantify the degree of under- or over-expression of a protein from two reciprocal experiments, a mutant p53 association index (mPAI) was defined as the log (of base 2) ratio of two readings from the two reciprocal experiments. Values of mPAI were normally distributed with a mean close to zero, consistent with the fact that levels of the majority of proteins were not affected by mtp53 knockdown. Standard deviation of mPAI is close to one and mtp53 itself shows mPAI values of greater than 2.0 (z-score > 2.0) in both cytosol and chromatin fractions, consistent with the expectation that its levels are significantly reduced by knockdown experiments. We thereby identified proteins and pathways significantly affected by mtp53 knockdowns as those with mPAI > 1.0 (indicating positive association) or mPAI Citation Format: Wei-Gang Qiu, Alla Polotskaia, Gu Xiao, Lia Di, John Philip, Ronald C. Hendrikson, Jill Bargonetti. Proteome-wide triple negative breast cancer mutant p53 association index identifies chromatin unwinding for precision therapeutics. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3684.

Abstract 1225: Proteome-wide analysis of gain-of-function mutant p53 targets in breast cancer implicates PARP, PCNA and MCM4 as oncogenic drivers

Gain-of-function mutant p53 (mtp53) changes the cancer cell transcriptome. However no analysis of mtp53-associated proteome diversity has been carried out. We coupled cell fractionation with stable isotope labeling with amino acids in cell culture (SILAC) and inducible knockdown of endogenous mtp53 to determine the mtp53 driven proteome. Using SILAC followed by tandem mass spectrometry (LC-MS/MS) we identified that R273H mtp53 expression in MDA-MB-468 breast cancer cells both up and down-regulated multiple proteins and metabolic pathways. We sequenced 73,154 peptide pairs that corresponded to 3010 proteins detected under reciprocal labeling conditions. Importantly the high impact regulated targets included the previously identified transcriptionally regulated mevalonate pathway proteins but also identified two new levels of mtp53 protein regulation for non-transcriptional targets. Interestingly, mtp53 depletion profoundly influenced poly (ADP-ribose) polymerase 1 (PARP1) localization, with increased cytoplasmic and decreased chromatin-associated PARP1. An enzymatic PARP shift occurred with high mtp53 expression resulting in increased poly-ADP-ribosylated proteins in the nucleus. Mtp53 increased the level of PCNA and MCM4 protein without changing the amount of pcna or mcm4 transcript. Pathway enrichment analysis ranked the DNA replication pathway above the cholesterol biosynthesis pathway as an R273H mtp53 activated proteomic target. Knowledge of the proteome diversity driven by mtp53 suggests DNA replication and repair pathways are major targets of mtp53 and highlights consideration of combination chemotherapeutic strategies targeting cholesterol biosynthesis and PARP inhibition. Acknowledgements. This work was supported by a grant to JB from the Breast Cancer Research Foundation. Citation Format: Alla Polotskaia, Gu Xiao, Katherine Reynoso, Che Martin, Wei-Gang Qiu, Ronald Hendrickson, Jill Bargonetti. Proteome-wide analysis of gain-of-function mutant p53 targets in breast cancer implicates PARP, PCNA and MCM4 as oncogenic drivers. [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 1225. doi:10.1158/1538-7445.AM2015-1225