P53-mutant Breast Cancer Cells Research Articles

Benzimidazole carbamate induces cytotoxicity in breast cancer cells via two distinct cell death mechanisms

Metastatic breast cancer (mBC) is responsible for >90% of breast cancer-related deaths. Microtubule-targeting agents (MTAs) are the front-line treatment for mBC. However, the effectiveness of MTAs is frequently limited by the primary or acquired resistance. Furthermore, recurrent mBC derived from cancer cells that survived MTA treatment are typically more chemoresistant. The overall response rates for the second- and third-line MTAs in mBC patients previously treated with MTAs are 12–35%. Thus, there is an ongoing search for novel MTAs with a distinct mode of action that can circumvent chemoresistance mechanisms. Our results show that methyl N-(6-benzoyl-1H-benzimidazol-2-yl)carbamate (BCar), a microtubule-disrupting anthelmintic that binds to the colchicine binding site separate from the binding sites of clinically used MTAs, has the potential to treat MTA-resistant mBC. We have comprehensively evaluated the cellular effects of BCar in a panel of human breast cancer (BC) cell lines and normal breast cells. BCar effects on the clonogenic survival, cell cycle, apoptosis, autophagy, senescence, and mitotic catastrophe were measured. Approximately 25% of BCs harbor mutant p53. For this reason, the p53 status was included as a variable. The results show that BC cells are >10x more sensitive to BCar than normal mammary epithelial cells (HME). p53-mutant BC cells are significantly more sensitive to BCar treatment than p53 wild-type BC cells. Furthermore, BCar appears to kill BC cells primarily via either p53-dependent apoptosis or p53-independent mitotic catastrophe. When compared to docetaxel and vincristine, two clinical MTAs, BCar is fairly innocuous in HME cells, providing a much wider therapeutic window than docetaxel and vincristine. Together, the results strongly support the notion that BCar-based therapeutics may serve as a new line of MTAs for mBC treatment.

Upregulation of sphingosine kinase 1 in response to doxorubicin generates an angiogenic response via stabilization of Snail.

Sphingosine kinase 1 (SK1) converts the pro-death lipid sphingosine to the pro-survival sphingosine-1-phosphate (S1P) and is upregulated in several cancers. DNA damaging agents, such as the chemotherapeutic doxorubicin (Dox), have been shown to degrade SK1 protein in cancer cells, a process dependent on wild-type p53. As mutations in p53 are very common across several types of cancer, we evaluated the effects of Dox on SK1 in p53 mutant cancer cells. In the p53 mutant breast cancer cell line MDA-MB-231, we show that Dox treatment significantly increases SK1 protein and S1P. Using MDA-MB-231 cells with CRISPR-mediated knockout of SK1 or the selective SK1 inhibitor PF-543, we implicated SK1 in both Dox-induced migration and in a newly uncovered proangiogenic program induced by Dox. Mechanistically, inhibition of SK1 suppressed the induction of the cytokine BMP4 and of the EMT transcription factor Snail in response to Dox. Interestingly, induction of BMP4 by SK1 increased Snail levels following Dox treatment by stabilizing Snail protein. Furthermore, we found that SK1 was required for Dox-induced p38 MAP kinase phosphorylation and that active p38 MAPK in turn upregulated BMP4 and Snail, positioning p38 downstream of SK1 and upstream of BMP4/Snail. Modulating production of S1P by inhibition of de novo sphingolipid synthesis or knockdown of the S1P-degrading enzyme S1P lyase identified S1P as the sphingolipid activator of p38 in this model. This work establishes a novel angiogenic pathway in response to a commonly utilized chemotherapeutic and highlights the potential of SK1 as a secondary drug target for patients with p53 mutant cancer.

549 Temporary cell cycle arrest in human scalp hair follicles and their epithelial stem cells by ALRN-6924: A novel strategy to selectively protect p53-wildtype cells against paclitaxel-induced alopecia

Chemotherapy-induced alopecia (CIA) is a highly distressing side effect of cancer therapy, and taxanes like paclitaxel (PTX) cause severe and often permanent CIA. Yet, strategies that selectively protect healthy cells, but not cancer cells, remain elusive. Here, we turned to ALRN-6924, a new clinical-stage MDM2/MDMX dual inhibitor that activates p53 to upregulate p21 and thus transiently arrests cell cycle in p53-wild-type healthy cells, but not p53-mutated cancer cells. Indeed, ALRN-6924 protects human fibroblasts (HS68) from multiple chemotherapies in vitro, but not p53-mutant breast cancer cells (BT-474).

Abstract GS1-09: Inhibition of GPX4 induces preferential death of p53-mutant triple-negative breast cancer cells

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype characterized by lack of ER, PR, and Her2. Up to 85% of TNBCs have a p53 mutation as their oncogenic driver. TP53 is a tumor suppressor known as the “guardian of the genome” for its roles in regulating growth and death after genomic insult. Due to this high frequency of p53 mutations in TNBCs, targeting p53 mutants in a clinical setting is highly attractive. However, there are currently no FDA-approved drugs that can directly target p53 mutant TNBCs. We propose pathways exist that, when inhibited, will induce the specific death of p53-mutant breast cancer cells, but not p53-wild type breast cells. To investigate this hypothesis, we performed high-throughput drug screening to identify drugs that induce death in p53-mutant TNBCs. We then characterized the identified drugs for mechanism of death induction and in vivo drug effect. Methods: In vitro and in silico drug screens were conducted with small-molecule libraries of drugs with known protein targets, and screens were integrated and common drugs identified. P53-wild type and mutant cells were treated with identified drugs and stained with DAPI and DRAQ7 to identify growth-suppressive and death-inductive effects. One of these drugs, the GPX4 inhibitor ML-162, was selected for further study. Mechanism of death induction by ML-162 was determined with AnnexinV/PI, caspase cleavage, and ferroptosis assays. To confirm these results, GPX4 was knocked out and replicated the effect of ML-162. To determine the reliance of this phenotype on p53 mutational status, a series of inducible p53-mutant cell lines were created in ER+ and TNBC cell lines and then treated with small molecule inhibitors. ML-162 in vivo effect was demonstrated by treating p53-mutant TNBC xenografts in nude mice. Results: Integration of in vitro and in silico screens identified 6 common drugs, representing cell cycle inhibitors, cell division inhibitors, a proteasome inhibitor, and a peroxidase inhibitor. Identified drugs were demonstrated to reduce growth or induce death of p53-mutant TNBC cell lines. The GPX4 inhibitor ML-162 was further characterized, and found to induce death through ferroptosis, and not apoptosis or necroptosis. GPX4 knockout in p53-mutant TNBC cell lines induced ferroptosis and can be blocked with an anti-ferroptosis drug. Inducible p53-mutations in ER+ and TNBC cell lines were treated with ML-162. P53 mutations in TNBC, and not ER+, cell lines showed increased sensitivity to ML-162. To demonstrate in vivo effect of ML-162, TNBC cell line xenografts were grown in nude mice and treated with ML-162. This treatment significantly reduced tumor volume and also induced lipid peroxidation, a hallmark of ferroptosis. Conclusion: Our high-throughput screening demonstrated several of the identified drugs suppress growth or induce death preferentially in p53-mutant breast cancers. One of these drugs, ML-162, induces death of p53-mutant triple-negative breast cancer cells through induction of ferroptosis, both in vitro and in vivo. These studies provide the basic science foundation to further develop ferroptosis inducers for the targeted treatment of p53-mutant breast cancers. This work was funded by the John Charles Cain Endowment. Citation Format: William M Tahaney, Jing Qian, Reid Powell, Cassandra L Moyer, Yanxia Ma, Nghi Nguyen, Jamal Hill, Clifford Stephan, Abhijit Mazumdar, Peter JA Davies, Powel H Brown. Inhibition of GPX4 induces preferential death of p53-mutant triple-negative breast cancer cells [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr GS1-09.

Dual inhibition of DNA-PK and DNA polymerase theta overcomes radiation resistance induced by p53 deficiency.

TP53 deficiency in cancer is associated with poor patient outcomes and resistance to DNA damaging therapies. However, the mechanisms underlying treatment resistance in p53-deficient cells remain poorly characterized. Using live cell imaging of DNA double-strand breaks (DSBs) and cell cycle state transitions, we show that p53-deficient cells exhibit accelerated repair of radiomimetic-induced DSBs arising in S phase. Low-dose DNA-dependent protein kinase (DNA-PK) inhibition increases the S-phase DSB burden in p53-deficient cells, resulting in elevated rates of mitotic catastrophe. However, a subset of p53-deficient cells exhibits intrinsic resistance to radiomimetic-induced DSBs despite DNA-PK inhibition. We show that p53-deficient cells under DNA-PK inhibition utilize DNA polymerase theta (Pol θ)-mediated end joining repair to promote their viability in response to therapy-induced DSBs. Pol θ inhibition selectively increases S-phase DSB burden after radiomimetic therapy and promotes prolonged G2 arrest. Dual inhibition of DNA-PK and Pol θ restores radiation sensitivity in p53-deficient cells as well as in p53-mutant breast cancer cell lines. Thus, combination targeting of DNA-PK- and Pol θ-dependent end joining repair represents a promising strategy for overcoming resistance to DNA damaging therapies in p53-deficient cancers.

Dendrosomal Nano-Curcumin Modulates P-Glycoprotein Activity and Induces Apoptosis in Wild Type and P53-Mutant Breast Cancer Cell Lines

Background: Dendrosomal nano-curcumin (DNC) represents an enormous potential to serve as a therapeutic anticancer agent. Here, we investigated the effect of DNC on wild type- and p53-mutant breast cancer cells. Methods: MCF-7 and T47D cells were treated with DNC and investigated for cell viability through MTT assay. The mode of death was analyzed using Annexin V/FITC staining and PARP cleavage assays. Flow cytometric efflux and cell swelling tests were employed to assess the p-glycoprotein (P-gp) transporter activity. Moreover, real-time PCR was employed to study the expression of Bax, Bcl-2 as well as survivin and its ΔEx3 splicing variant. Results: Our findings confirmed that DNC repressed cancer cell proliferation by induction of apoptosis. Additionally, DNC significantly modulated P-gp function that might lead to improved cellular permanence of curcumin. Malfunction of P-gp activity by DNC demonstrates its capability in reducing the drug resistance of p53-mutant cancer cells. Conclusions: In conclusion, our findings demonstrated that dendrosomal nano-curcumin could be considered an anti-tumor therapeutic for p53-mutant tumor malignancies.

UBR5 Is Coamplified with MYC in Breast Tumors and Encodes an Ubiquitin Ligase That Limits MYC-Dependent Apoptosis.

For maximal oncogenic activity, cellular MYC protein levels need to be tightly controlled so that they do not induce apoptosis. Here, we show how ubiquitin ligase UBR5 functions as a molecular rheostat to prevent excess accumulation of MYC protein. UBR5 ubiquitinates MYC and its effects on MYC protein stability are independent of FBXW7. Silencing of endogenous UBR5 induced MYC protein expression and regulated MYC target genes. Consistent with the tumor suppressor function of UBR5 (HYD) in Drosophila, HYD suppressed dMYC-dependent overgrowth of wing imaginal discs. In contrast, in cancer cells, UBR5 suppressed MYC-dependent priming to therapy-induced apoptosis. Of direct cancer relevance, MYC and UBR5 genes were coamplified in MYC-driven human cancers. Functionally, UBR5 suppressed MYC-mediated apoptosis in p53-mutant breast cancer cells with UBR5/MYC coamplification. Furthermore, single-cell immunofluorescence analysis demonstrated reciprocal expression of UBR5 and MYC in human basal-type breast cancer tissues. In summary, UBR5 is a novel MYC ubiquitin ligase and an endogenous rheostat for MYC activity. In MYC-amplified, and p53-mutant breast cancer cells, UBR5 has an important role in suppressing MYC-mediated apoptosis priming and in protection from drug-induced apoptosis. SIGNIFICANCE: These findings identify UBR5 as a novel MYC regulator, the inactivation of which could be very important for understanding of MYC dysregulation on cancer cells. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/7/1414/F1.large.jpg.

A novel series of nitrofuran derivatives produces an anti‐tumor effect via a p53‐dependent mechanism

Nitrofurantoin (NF) is an antibacterial that acts by inducing DNA damage. Increased selectivity towards bacterial DNA is conferred through the rapid activation by prokaryotic reductases. In cancer therapy, radiation and anti‐neoplastic drugs induce DNA damage to activate the p53‐dependent pathway, provoking apoptosis in cancer cells. In this study, a novel series of nitrofuran derivatives was designed with a presumed enhanced eukaryotic activity. The safety and the anti‐tumor efficacy of this series was assessed. Anticancer effects were assessed on colorectal cancer (Caco‐2), breast cancer (MCF‐7), cervical cancer (Hela), and liver cancer cells (HepG‐2). Cytotoxicity was estimated by MTT assays. Three compounds, NF‐5, NPO, and NS‐1 were shown to have the highest cytotoxic activity against cancer cells at IC50 values that are several‐fold lower that those observed for non‐malignant lung fibroblasts. The abilities to induce oxidative stress and apoptosis were tested by flow cytometry to measure dihydrodichlorofluorescein fluorescence and nuclear annexin‐V staining, and compared to that of NF and the reference chemotherapeutic drug (5‐FU). Other apoptosis markers measured include caspase3/7 activity and Bax/Bcl2 ratio. NF‐5, NPO, and NS‐1 induced oxidative DNA damage in cancer cells in vitro with subsequent cellular apoptosis. Furthermore, the three derivatives induced more than 50% increase in reactive oxygen species when compared to 5‐FU. Cells treated with these drugs showed an increased Bax/Bcl2 ratio and caspase 3/7 activity compared to the traditional chemotherapeutic drug. A positive linear correlation was shown between the oxidative load produced in the cells and the increase in apoptotic markers. Interestingly, despite equivalent induction of DNA damage demonstrated by similar pATM nuclear shuttling, NF‐5, NPO, and NS‐1 produced more cytotoxicity in MCF‐7 cells compared to the p53 mutant breast cancer cell line MDA‐MB‐231. Moreover, the effect of these derivatives on the p53 interaction with its negative modulator Murine Double Minute 2 (MDM2) was assessed using a multi‐spot sandwich ELISA kit. NF‐5, NPO, and NS‐1 promoted p53 release from MDM2 at concentrations that are 2–3 orders of magnitude lower than their cytotoxic IC50 values. These findings were further confirmed by in silico docking studies demonstrating possible binding sites on the protein. Further experiments are underway to assess the ability of lower, non‐cytotoxic concentrations of these drugs to improve the radio‐sensitivity of cancer cells via activation of p53‐dependnet pathways.Support or Funding InformationFunded by: AUB‐FM MPP grant #320148

Abstract P3-03-08: Identification of drugs that induce the death or suppress the growth of p53-mutant breast cancer

Abstract Background: P53 is the most commonly mutated protein in cancer with more than 40% of all cancer patients harboring a p53 mutation. Up to 85% of triple-negative breast cancers (TNBCs), aggressive breast cancers characterized by the absence of ER, PR, and Her2 receptors, have p53 mutations. P53 is a tumor suppressor that helps balance cellular growth and death after a genomic insult. Due to the rate of p53 mutation in cancer, the targeting of p53 in a clinical setting is an attractive option. However, currently there are no available p53-targeted drugs. We hypothesized that molecular pathways exist, that when inhibited, induce death or suppress growth of p53-mutant breast cancer cells, without affecting p53 wild-type breast cells. To investigate this hypothesis, we conducted a high-throughput drug screen to identify drugs that induce death or suppress growth of p53-mutant TNBCs. Methods: A library of 453 drugs with diverse and known mechanisms of action was used to perform high-throughput drug screening through collaboration with Texas A&M’s Center for Translational Cancer Research. An ATP luminescence assay was used to test eight TNBC cell lines with distinct p53 mutations in this screen. We then tested a subset of these drugs, identified to have an effect on p53-mutant TNBC, on the growth of five p53 wild-type breast cancer cell lines. Cell death assays to differentiate between growth suppression and death-inductive phenotypes are currently underway. Results: 145 drugs were identified that induce death or decrease growth by a minimum of 50% in p53-mutant TNBC cell lines. Of these drugs, 16 drugs were found to differentially suppress the growth of p53-mutant breast cancer cells, as compared to p53-wild type breast cancer cells. Further analysis of these drugs identified proteasome, apoptosis, and iron-dependent cell death pathways as potential p53-mutant specific targets in TNBCs. Conclusion: A screen of molecularly annotated drugs was performed in p53-mutant and p53-wild type breast cancer cells to identify drugs that preferentially kill or suppress the growth of p53-mutant breast cancers. These studies identified 145 drugs that were able to induce death or decrease growth by a minimum of 50% in p53-mutant TNBC cell lines. Testing these drugs in p53 wild-type breast cancer cell lines demonstrated that several of the identified drugs suppress growth or induce death preferentially in p53-mutant, as compared to p53 wild-type, breast cancers. These drugs affect specific cellular pathways such as proteasomal degradation, ferroptosis, and apoptosis pathways, which appear to be critical for the growth and survival of p53-mutant breast cancers. Further work will elucidate the interplay between p53-mutation and the identified cellular pathways. These studies will provide the basic science foundation for the development of new drugs for the treatment of p53-mutant breast cancer. This work was supported by a generous gift from the John Charles Cain Family. Citation Format: William Miska Tahaney, Reid Powell, Nghi Nguyen, Lakshmi Reddy Bollu, Jing Qian, Abhijit Mazumdar, Clifford Stephan, Peter JA Davies, Powel H Brown. Identification of drugs that induce the death or suppress the growth of p53-mutant breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-03-08.

Abstract 1218: Synthetic lethal targeting of p53 mutant cells with prenylation inhibitors

Abstract Introduction: TP53 is mutated in 30-40% of breast cancers and is associated with poor prognosis. Mutation of TP53 causes increased cellular proliferation, migration and invasion, and downstream activation of multiple pathways. HMG CoA reductase inhibitors, such as simvastatin, inhibit tumorigenic properties induced by TP53 mutation. Mechanism of this response is an important question in targeted cancer therapy. It is well known that HMG CoA reductase inhibitors block cholesterol production and prenylation. Therefore, we hypothesized that prenylation inhibitors would target p53 mutant cells primarily by inhibiting activation of the ras family induced by TP53 mutation. Method: We generated 5 MCF10A stably transduced cell lines over-expressing each of the common TP53 point mutations, R273H, G245S, R248Q, Y234C, or wild type p53. Total Ras, Ras-GTP, total RhoA and RhoA-GTP was measured by immunoprecipitation and immunoblot. Proliferation was measured using CellTiterGlo. The functional effects of a panel of prenylation inhibitors was measured using a caspase3 reporter assay, and migration was measured with a scratch assay. Illumina mRNA sequencing was performed to measure gene expression of mutant and wild type cells before and after simvastatin treatment (2.5µl, 48 h). The RNAseq was analyzed with the DAVID Enrichment tool to evaluate ras pathway activation induced by TP53 mutation. Results: Mutation in TP53 was associated with significant activation of both wild-type Ras and RhoA. Proliferation of cells expressing mutant TP53 was sensitive to a panel of prenylation inhibitors, including simvastatin. Growth inhibition by simvastatin correlated with induction of apoptosis, and could be fully rescued by addition of farnesylpyrophosphate or geranylgeranylpyrophosphate, suggesting that simvastatin functionally blocks both prenylation pathways which are activated in the presence of mutant p53 (IC50(wt/mut) = 4). RNAseq analysis confirms that Ras signaling pathways, including GAPs and GEFs are enriched in the presence of mutant TP53, and FOXO signaling is significantly targeted post statin treatment (P<0.01). Conclusion: p53 mutant breast cancer cells are highly sensitive to prenylation inhibition due to activation of both ras and rhoA. Simvastatin inhibits both farnesylation and geranylgeranylation, effectively blocking ras pathway activation and induction of proliferation in p53 mutant cell lines. A gene expression panel associated with ras pathway activation was identified and may be predictive biomarkers for sensitivity to statin therapy. Citation Format: Shay R. Ferdosi, Benjamin Katchman, Jia Loo, Harneet Grewal, Seron Eaton, Shanshan Yang, Jin Park, Joshua Labaer, Karen S. Anderson. Synthetic lethal targeting of p53 mutant cells with prenylation inhibitors [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 1218. doi:10.1158/1538-7445.AM2017-1218

Abstract 3526: The expression of MELK, a growth regulator of triple-negative breast cancer, is regulated by p53

Abstract Background: Triple negative breast cancer (TNBC) is the most aggressive form of breast cancer with poor prognosis. Due to frequent distant metastasis and lack of successful targeted therapies, the overall survival rate in TNBC patients is significantly lower than estrogen receptor positive (ER) and HER2 positive breast cancers. To identify potential druggable targets, previously, we performed a gene expression analysis and identified protein kinases that are highly expressed in TNBC. Through these studies, we discovered that the expression of maternal embryonic leucine zipper kinase (MELK) is highly elevated in TNBCs and correlated with p53-mutation status and metastasis-free survival. Inhibition of MELK reduced the growth and invasiveness of p53 mutant breast cancer cells. Methods: In this study, we analyzed the association of MELK expression with p53 status using publicly available datasets, and examined the role of wild-type and mutant P53 in regulating MELK expression. MELK expression at mRNA and protein levels were determined through Q-RT-PCR and western blotting analysis respectively. The association of p53 and MELK expression was determined using Oncomine analysis. We also investigated the effect of p53 knockdown and overexpression on MELK expressing using Q-RT-PCR, western blotting and promoter luciferase assays. Results: Analysis of publicly available datasets revealed that MELK expression is highly elevated p53-mutant breast cancer independent of estrogen receptor status. Consistent with observation, high MELK expression was found in p53-mutant cells compared to p53 wild-type cells. Through luciferase, western blotting and Q-RT-PCR assays, we discovered that the increased expression of MELK in TNBC cells is due to inactivation of p53 wild-type function. Consistently, over expression of p53 wild-type in p53-Null cells, using doxycycline inducible system reduced MELK promoter activity and expression. Similarly, inhibition of p53 wild-type by over expression of p53-mutants in p53-wild-type cells also increased MELK expression through dominant-negative effect. In contrast, knockdown of p53-mutants or over expression of p53-mutants in p53-null cells did not alter MELK expression. Finally, through promoter deletion studies coupled with luciferase studies, we identified a potential p53-response region in MELK promoter. Conclusion: Our results suggest that the expression of MELK, a critical regulator of growth and invasion of TNBCs, is regulated by the tumor suppressor gene, p53 wild-type. Inactivation of p53 wild-type either gene deletion or mutations induces the expression of MELK. These results suggest that MELK is a promising target for the treatment of women with p53-mutant TNBC. This work was supported by Susan G Komen Promise Grant (PB, SH, GM), SAB Komen grant (PB) and Young Foundation grant (PB). Citation Format: Lakshmi Reddy Bollu, Powel H. Brown, Abhijit Mazumdar, Dekuang Zhao, Yanxia Ma. The expression of MELK, a growth regulator of triple-negative breast cancer, is regulated by p53 [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 3526. doi:10.1158/1538-7445.AM2017-3526

Wild-type p53 controls the level of fibronectin expression in breast cancer cells.

Aberrant fibronectin (FN) expression is associated with poor prognosis, cell adhesion, and cell motility in a variety of cancer cells. In this study, we investigated the relationship between p53 and FN expression in breast cancer cells. Basal FN expression was significantly decreased by treatment with the p53 activator III, RITA, in MCF7 breast cancer cells with wild-type p53. In addition, overexpression of wild-type p53 markedly decreased the level of FN expression in p53-mutant breast cancer cells. To examine the mechanism underlying the relationship between p53 and FN expression, we treated MCF7 breast cancer cells with the tumor promoter TPA (12-O-tetradecanoylphorbol-13-acetate). Our results showed that basal FN expression was increased by TPA treatment in a time-dependent manner. In contrast, the level of p53 expression was decreased by TPA treatment. However, the expression of FN and p53 was not altered by TPA in p53-mutant breast cancer cells. Furthermore, the alterations in FN and p53 expression in response to TPA were prevented by a specific MEK inhibitor, UO126. Finally, we demonstrated that TPA triggers degradation of p53 through the proteasomal pathway in MCF7 cells. TPA-induced FN expression was decreased by the proteasome inhibitor MG132. Under the same condition, p53 protein expression, but not mRNA expression, was reversed by MG132. Taken together, our data demonstrate that the level of FN expression is associated with the status and expression of p53 in breast cancer cells.

Tristetraprolin mediates the anti-proliferative effects of metformin in breast cancer cells.

Metformin, which is a drug commonly prescribed to treat type 2 diabetes, has anti-proliferative effects in cancer cells; however, the molecular mechanisms underlying this effect remain largely unknown. The aim is to investigate the role of tristetraprolin (TTP), an AU-rich element-binding protein, in anti-proliferative effects of metformin in cancer cells. p53 wild-type and p53 mutant breast cancer cells were treated with metformin, and expression of TTP and c-Myc was analyzed by semi-quantitative RT-PCR, Western blots, and promoter activity assay. Breast cancer cells were transfected with siRNA against TTP to inhibit TTP expression or c-Myc and, after metformin treatment, analyzed for cell proliferation by MTS assay. Metformin induces the expression of tristetraprolin (TTP) in breast cancer cells in a p53-independent manner. Importantly, inhibition of TTP abrogated the anti-proliferation effect of metformin. We observed that metformin decreased c-Myc levels, and ectopic expression of c-Myc blocked the effect of metformin on TTP expression and cell proliferation. Our data indicate that metformin induces TTP expression by reducing the expression of c-Myc, suggesting a new model whereby TTP acts as a mediator of metformin’s anti-proliferative activity in cancer cells.

Abstract 2329: DAPK-1, a death signaling transducer, is critical for the tumorigenicity of p53 mutant breast cancer.

Abstract Background: Breast cancers can be subgrouped into ER-positive and ER-negative breast cancers according to their estrogen receptor status. Few effective targeted treatments are available for the more aggressive ER-negative breast cancers, 80% of which carry p53 mutation. Our lab conducted a human kinome study which identified 52 kinases highly expressed in ER-negative breast tumors. One highly expressed kinase, DAPK-1, was selected for further study. Death associated protein kinase-1 (DAPK-1) transduces death signaling through P53-dependent or -independent pathways. In this study, we determined a novel role of DAPK-1 in tumorigenesis of ER-negative, p53 mutant breast cancers. Methods: To perform expression and survival analysis of DAPK-1 in breast tumors, we used data from VandeVijver, Desmedt and Ivshina datasets. Inducible DAPK-1 knockdown cell lines were constructed through pTRIPZ lentiviral system. Anchorage-independent growth was performed in soft agar and colonies were enumerated after 15-30 days. Xenograft experiments were performed in nude mice. Proteomic analysis was used to determine downstream signaling. Results: Gene expression analysis showed that high DAPK-1 expression correlates significantly with the ER-negative subgroup of breast cancer. Using an inducible knockdown system, we found that depletion of DAPK-1 strongly suppressed in vitro growth and in vivo tumorigenesis of ER-negative, P53-mutant cells. Through a siRNA knockdown study in an expanded panel of breast cancer cell lines, we established an association between p53 mutation status and sensitivity of the cells to DAPK-1 depletion. ER-negative breast cancer cell lines with P53 mutations are very sensitive to DAPK-1 knockdown, while ER-positive breast cancer cell lines with wild-type P53 are resistant to DAPK-1 knockdown. By depleting P53 in P53 wild-type cells, we then showed that P53 wild-type breast cancer cells become partially dependent on DAPK-1 upon P53 dysfunction. Through proteomic analysis, we determined that DAPK-1 regulates cell growth through phosphorylating TSC2 at Ser939 and interfering with TSC1/TSC2 complex, which is a well-known repressor of mTOR pathway. Our survival analysis confirmed that high DAPK-1 expression in breast tumors is associated with worse clinical outcome, especially in patients that carry P53 mutations. Conclusion: P53-mutant breast cancer cells are sensitive to DAPK-1 inhibition in vitro and in vivo. P53 dysfunction causes breast cancer cells to become dependent on DAPK-1 signaling for their growth. High expression of DAPK-1 in P53-mutant breast tumor activates the mTOR pathway through relieving the TSC1/TSC2-mediated repressor of mTOR signaling. Our results demonstrate a dual role of DAPK-1 in regulating breast cancer proliferation and apoptosis depending on the P53 status. This study is supported by a PROMISE grant (KG081694) from Susan G. Komen for the Cure Foundation. Citation Format: Jing Zhao, Yun Zhang, Jamal L. Hill, Zack Hartman, Abhijit Mazumdar, Gordon B. Mills, Powel H. Brown. DAPK-1, a death signaling transducer, is critical for the tumorigenicity of p53 mutant breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2329. doi:10.1158/1538-7445.AM2013-2329

Mitotic Arrest and Apoptosis in Breast Cancer Cells Induced by Origanum majorana Extract: Upregulation of TNF-α and Downregulation of Survivin and Mutant p53

BackgroundIn the present study, we investigated the effect of Origanum majorana ethanolic extract on the survival of the highly proliferative and invasive triple-negative p53 mutant breast cancer cell line MDA-MB-231.ResultsWe found that O. majorana extract (OME) was able to inhibit the viability of the MDA-MB-231 cells in a time- and concentration-dependent manner. The effect of OME on cellular viability was further confirmed by the inhibition of colony growth. We showed, depending on the concentration used, that OME elicited different effects on the MDA-MB 231 cells. Concentrations of 150 and 300 µg/mL induced an accumulation of apoptotic–resistant population of cells arrested in mitotis and overexpressing the cyclin-dependent kinase inhibitor, p21 and the inhibitor of apoptosis, survivin. On the other hand, higher concentrations of OME (450 and 600 µg/mL) triggered a massive apoptosis through the extrinsic pathway, including the activation of tumor necrosis factor-α (TNF-α), caspase 8, caspase 3, and cleavage of PARP, downregulation of survivin as well as depletion of the mutant p53 in MDA-MB-231 cells. Furthermore, OME induced an upregulation of γ-H2AX, a marker of double strand DNA breaks and an overall histone H3 and H4 hyperacetylation.ConclusionOur findings provide strong evidence that O. majorana may be a promising chemopreventive and therapeutic candidate against cancer especially for highly invasive triple negative p53 mutant breast cancer; thus validating its complementary and alternative medicinal use.

The Chk1 inhibitor AZD7762 sensitises p53 mutant breast cancer cells to radiation in vitro and in vivo

AZD7762, a novel checkpoint kinase 1 (Chk 1)inhibitor, has been proven to sensitize various tumor cells to DNA damage. However, whether or not AZD7762 sensitizes breast cancer cells to radiation has not been defined. In the present study, we aimed to demonstrate for the first time, that AZD7762 not only promotes radiation-induced apoptosis and mitotic catastrophe of p53 mutant T47D breast cancer cells in vitro, but also delays their xenograft growth in response to radiation in vivo. Our mechanistic study showed that AZD7762 treatment resulted in the abrogation of radiation-induced G2/M arrest and the inhibition of radiation damage repair as demonstrated by increased radiation-induced γH2AX expression and decreased RAD51 protein expression. These results suggest that AZD7762 may effectively abrogate radiation-induced G2/M arrest and inhibit radiation damage repair in conferring radiosensitivity on p53 mutant T47D breast cancer cells, by promoting radiation-induced apoptosis and mitotic catastrophe. The clinical application of AZD7762, as an adjuvant in the radiotherapy of breast cancers, should be further explored.

Increased Expression of the E3 Ubiquitin Ligase RNF5 Is Associated with Decreased Survival in Breast Cancer

The selective ubiquitination of proteins by ubiquitin E3 ligases plays an important regulatory role in control of cell differentiation, growth, and transformation and their dysregulation is often associated with pathologic outcomes, including tumorigenesis. RNF5 is an E3 ubiquitin ligase that has been implicated in motility and endoplasmic reticulum stress response. Here, we show that RNF5 expression is up-regulated in breast cancer tumors and related cell lines. Elevated expression of RNF5 was seen in breast cancer cell lines that became more sensitive to cytochalasin D- and paclitaxel-induced apoptosis following its knockdown with specific short interfering RNA. Inhibition of RNF5 expression markedly decreased cell proliferation and caused a reorganization of the actin cytoskeleton in response to stress in MCF-7 but not in p53 mutant breast cancer cells, suggesting a p53-dependent function. Significantly, high levels of RNF5 were associated with decreased survival in human breast cancer specimens. Similarly, RNF5 levels were higher in metastatic melanoma specimens and in melanoma, leukemia, ovarian, and renal tumor-derived cell lines, suggesting that increased RNF5 expression may be a common event during tumor progression. These results indicate that RNF5 is a novel regulator of breast cancer progression through its effect on actin cytoskeletal alterations, which also affect sensitivity of breast cancer cells to cytoskeletal targeting antineoplastic agents.

HOXA5-induced apoptosis in breast cancer cells is mediated by caspases 2 and 8.

HOXA5 is a transcriptional factor whose expression is lost in more than 60% of breast carcinomas. Our previous work demonstrated that the overexpression of HOXA5 in MCF7 cells resulted in cell death through a p53-dependent apoptotic pathway. To determine whether p53-independent apoptotic pathways are involved in HOXA5-induced cell death, we engineered a p53-mutant breast cancer cell line, Hs578T, to inducibly express HOXA5. Induction of HOXA5 expression led to cell death with features typical of apoptosis within 24 h, and the expression levels of mutant p53 and its target genes either decreased or remained unchanged. To decipher apoptotic pathways, the HOXA5-expressing cells were treated with a variety of apoptotic inhibitors. Besides a general caspase inhibitor, caspase 2- and 8-specific inhibitors largely abolished HOXA5-induced apoptosis, whereas caspase 1-, 3-, 6-, and 9-specific inhibitors had no significant effects. Western blot analysis further confirmed that caspases 2 and 8 were activated after the induction of HOXA5 expression. Further, several small interfering RNAs which specifically silenced caspase 2 and caspase 8 expression significantly blocked HOXA5-induced apoptosis. HOXA5 expression could also sensitize cells to tumor necrosis factor alpha-induced apoptosis by at least 100-fold. These results indicate that expression of HOXA5 can induce apoptosis through an apoptotic mechanism mediated by caspases 2 and 8.