Alla Polotskaia

A p53-independent role of Mdm2 in estrogen-mediated activation of breast cancer cell proliferation

IntroductionEstrogen receptor positive breast cancers often have high levels of Mdm2. We investigated if estrogen signaling in such breast cancers occurred through an Mdm2 mediated pathway with subsequent inactivation of p53.MethodsWe examined the effect of long-term 17β-estradiol (E2) treatment (five days) on the p53-Mdm2 pathway in estrogen receptor alpha (ERα) positive breast cancer cell lines that contain wild-type p53 (MCF-7 and ZR75-1). We assessed the influence of estrogen by examining cell proliferation changes, activation of transcription of p53 target genes, p53-chromatin interactions and cell cycle profile changes. To determine the effects of Mdm2 and p53 knockdown on the estrogen-mediated proliferation signals we generated MCF-7 cell lines with inducible shRNA for mdm2 or p53 and monitored their influence on estrogen-mediated outcomes. To further address the p53-independent effect of Mdm2 in ERα positive breast cancer we generated cell lines with inducible shRNA to mdm2 using the mutant p53 expressing cell line T-47D.ResultsEstrogen increased the Mdm2 protein level in MCF-7 cells without decreasing the p53 protein level. After estrogen treatment of MCF-7 cells, down-regulation of basal transcription of p53 target genes puma and p21 was observed. Estrogen treatment also down-regulated etoposide activated transcription of puma, but not p21. Mdm2 knockdown in MCF-7 cells increased p21 mRNA and protein, decreased cell growth in 3D matrigel and also decreased estrogen-induced cell proliferation in 2D culture. In contrast, knockdown of p53 had no effect on estrogen-induced cell proliferation. In T-47D cells with mutant p53, the knockdown of Mdm2 decreased estrogen-mediated cell proliferation but did not increase p21 protein.ConclusionsEstrogen-induced breast cancer cell proliferation required a p53-independent role of Mdm2. The combined influence of genetic and environmental factors on the tumor promoting effects of estrogen implicated Mdm2 as a strong contributor to the bypass of cell cycle checkpoints. The novel finding that p53 was not the key target of Mdm2 in the estrogen activation of cell proliferation could have great benefit for future Mdm2-targeted breast cancer therapies.

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.

Endogenous Human MDM2-C Is Highly Expressed in Human Cancers and Functions as a p53-Independent Growth Activator

Human cancers over-expressing mdm2, through a T to G variation at a single nucleotide polymorphism at position 309 (mdm2 SNP309), have functionally inactivated p53 that is not effectively degraded. They also have high expression of the alternatively spliced transcript, mdm2-C. Alternatively spliced mdm2 transcripts are expressed in many forms of human cancer and when they are exogenously expressed they transform human cells. However no study to date has detected endogenous MDM2 protein isoforms. Studies with exogenous expression of splice variants have been carried out with mdm2-A and mdm2-B, but the mdm2-C isoform has remained virtually unexplored. We addressed the cellular influence of exogenously expressed MDM2-C, and asked if endogenous MDM2-C protein was present in human cancers. To detect endogenous MDM2-C protein, we created a human MDM2-C antibody to the splice junction epitope of exons four and ten (MDM2 C410) and validated the antibody with in vitro translated full length MDM2 compared to MDM2-C. Interestingly, we discovered that MDM2-C co-migrates with MDM2-FL at approximately 98 kDa. Using the validated C410 antibody, we detected high expression of endogenous MDM2-C in human cancer cell lines and human cancer tissues. In the estrogen receptor positive (ER+) mdm2 G/G SNP309 breast cancer cell line, T47D, we observed an increase in endogenous MDM2-C protein with estrogen treatment. MDM2-C localized to the nucleus and the cytoplasm. We examined the biological activity of MDM2-C by exogenously expressing the protein and observed that MDM2-C did not efficiently target p53 for degradation or reduce p53 transcriptional activity. Exogenous expression of MDM2-C in p53-null human cancer cells increased colony formation, indicating p53-independent tumorigenic properties. Our data indicate a role for MDM2-C that does not require the inhibition of p53 for increasing cancer cell proliferation and survival.

8-amino-adenosine Activates p53-Independent Cell Death of Metastatic Breast Cancers

8-Amino-adenosine (8-NH2-Ado) is a ribose sugar nucleoside analogue that reduces cellular ATP levels and inhibits mRNA synthesis. Estrogen receptor-negative (ER−) metastatic breast cancers often contain mutant p53; therefore, we asked if 8-NH2-Ado could kill breast cancer cells without activating the p53-pathway. Regardless of the breast cancer subtype tested or the p53 status of the cells, 8-NH2-Ado was more cytotoxic than either gemcitabine or etoposide. 8-NH2-Ado treatment inhibited cell proliferation, activated cell death, and did not activate transcription of the p53 target gene p21 or increase protein levels of either p53 or p21. This occurred in the estrogen receptor-positive (ER+) MCF-7 cells that express wild-type p53, the ER+ T47-D cells that express mutant p53, and the ER− MDA-MB-468 cells or MDA-MB-231 cells that both express mutant p53. 8-NH2-Ado induced apoptotic death of MCF-7 cells and apoptosis was not inhibited by knockdown of functional p53. Moreover, the pan-caspase inhibitor Z-VAD blocked the 8-NH2-Ado–induced MCF-7 cell death. Interestingly, 8-NH2-Ado caused the MDA-MB-231 cells to detach from the plate with only limited evidence of apoptotic cell death markers and the cell death was not inhibited by Z-VAD. Inhibition of MDA-MB-231 cell autophagy, by reduction of ATG7 or 3-methyladenine treatment, did not block this 8-NH2-Ado–mediated cytotoxicity. Importantly 8-NH2-Ado was highly cytotoxic to triple-negative breast cancer cells and worked through a pathway that did not require wild-type p53 for cytoxicity. Therefore, 8-NH2-Ado should be considered for the treatment of triple-negative breast cancers that are chemotherapy resistant. Mol Cancer Ther; 11(11); 2495–504. ©2012 AACR.

Impedimetric Detection of Mutant p53 Biomarker-Driven Metastatic Breast Cancers under Hyposmotic Pressure

In cancer cells, the oncogenic mutant p53 (mtp53) protein is present at high levels and gain-of-function (GOF) activities with more expression of mtp53 proteins contribute to tumor growth and metastasis. Robust analytical approaches that probe the degree of metastasis of cancer cells in connection with the mtp53 activity will be extremely useful not only for establishing a better cancer prognosis but also understanding the fundamental mechanism of mtp53 oncogenic action. Here we assessed the influence of mtp53 in breast cancers to the mechanical property of breast cancer cells. Recently, ovarian and kidney cancer cell lines have been shown to have higher cellular elasticity as compared to normal cells assessed by monitoring the degree of deformation under hyposmotic pressure. To make fast detection in large scale, the impedance measurement was applied to monitor the swelling ratio of cells with time. The results showed that knockdown of mtp53 leads to decrease in cell swelling. In addition, by means of two types of impedimetric detection systems we consistently detected enhancement of impedance signal in mtp53-expressing breast cancer cells. Based on this observation we hypothesize that highly expressed mtp53 in metastatic mutant breast cancers can promote tumor progression by making cells more deformable and easier to spread out through extracellular matrix. The identification via the electric measurement can be accomplished within 10 minutes. All results in this report suggest that electric probing for the extent of the mtp53 expression of breast cancer cells may serve as a meaningful fingerprint for the cancer diagnostics, and this outcome will also have an important clinical implication for the development of mtp53-based targeting for tumor detection and treatment.

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.

Hot Spot Mutation in TP53 (R248Q) Causes Oncogenic Gain-of-Function Phenotypes in a Breast Cancer Cell Line Derived from an African American patient

African American (AA) breast cancer patients often have triple negative breast cancer (TNBC) that contains mutations in the TP53 gene. The point mutations at amino acid residues R273 and R248 both result in oncogenic gain-of-function (GOF) phenotypes. Expression of mutant p53 (mtp53) R273H associates with increased cell elasticity, survival under serum deprivation conditions, and increased Poly (ADP ribose) polymerase 1 (PARP1) on the chromatin in the AA-derived TNBC breast cancer cell line MDA-MB-468. We hypothesized that GOF mtp53 R248Q expression could stimulate a similar phenotype in the AA-derived TNBC cell line HCC70. To test this hypothesis we depleted the R248Q protein in the HCC70 cell line using shRNA-mediated knockdown. Using impedance-based real-time analysis we correlated the expression of mtp53 R248Q with increased cell deformability. We also documented that depletion of mtp53 R248Q increased PARP1 in the cytoplasm and decreased PARP1 on the chromatin. We conclude that in the AA-derived TNBC HCC70 cells mtp53 R248Q expression results in a causative tumor associated phenotype. This study supports using the biological markers of high expression of mtp53 R273H or R248Q as additional diagnostics for TNBC resistant subtypes often found in the AA community. Each mtp53 protein must be considered separately and this work adds R248Q to the increasing list of p53 mutations that can be used for diagnostics and drug targeting. Here we report that when R248Q mtp53 proteins are expressed in TNBC, then targeting the gain-of-function pathways may improve treatment efficacy.

Correction: A p53-independent role of Mdm2 in estrogen-mediated activation of breast cancer cell proliferation

Following publication of our article, the authors noticed that the data incorporated into Figure 4f was incorrect. The first column should be changed from 44, 13 and 46 to read 43, 13 and 44 instead. A corrected version of Figure 4f can be found overleaf.

Abstract 1169: Non-canonical functions of Mdm2 isoforms in estrogen influenced breast cancer cells with wild-type or mutant p53

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Estrogen receptor positive breast cancers often have high levels of Mdm2. We investigated if estrogen signaling in breast cancers occurred through an Mdm2 mediated pathway. We examined the effect of long-term 17β-estradiol (E2) treatment in ERα positive breast cancer cell lines that contain wild-type p53 (MCF-7) and mutant p53 (T-47D). These cell lines also contain the mdm2 SNP at position 309, which increases the expression of Mdm2 by Sp1 driven transcription. In both these cell lines, we observed that E2 treatment dramatically increased the amount of Mdm2 without decreasing the corresponding wild-type or mutant p53. Surprising the amount of p53 protein in both cell lines increased with E2 treatment. We engineered miR30-based inducible mdm2 knockdown cell lines of MCF-7 and T-47D to evaluate to impact of Mdm2 on their cancer cell phenotypes. The down-regulation of Mdm2 in MCF-7.shmdm2 and T-47D.shmdm2 inhibited cell proliferation in 2D culture and in 3D laminin-rich matri-gel. Knockdown of Mdm2 did not cause an increase in p53 protein. These data suggest a p53-independent signal transduction pathway for Mdm2-mediated activation of proliferation. Alternative spliced mdm2 transcripts have been observed in many cancers including breast, but no endogenous protein isoforms from the spliced isoforms have been described. We asked if the spliced variant Mdm2-C transcript (that lacks most of the N-terminal p53 binding domain) expressed endogenous protein in these breast cancer cell lines. We created an Mdm2-C specific antibody using a synthetic peptide to the splice junction of exons four and ten (C4-10) to detect the protein. We validated the C4-10 antibody using in vitro translated Mdm2 compared to Mdm2-C. High expression of Mdm2-C occurred in the G/G mdm2 SNP309 ER+ breast cancer cell line T-47D, but only a slight increase was detected in the presence of E2. Additionally, Mdm2-C was found in nucleolar and cytoplasmic foci before and after estrogen treatment, while the mutant p53 was predominantly nuclear. We report the first identification of endogenous tumor-associated Mdm2-C protein in breast cancer. We suggest that Mdm2 and its isoforms work through non-canonical pathways to transform estrogen influenced breast cancers through molecular pathways that remain to be elucidated. Acknowledgements: This work was supported by Breast Cancer Research Foundation to J.B. and NSF grant to JB MCB-0744316. D.O. was also partially supported by a CUNY Graduate Center Magnet Award. Special thanks to Gerard Evan for help with immunogen peptide design. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1169. doi:1538-7445.AM2012-1169

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