P53 Wild-type Cancer Research Articles

MsrB3 deficiency induces cancer cell apoptosis through p53-independent and ER stress-dependent pathways

We have previously shown that down-regulation of methionine sulfoxide reductase B3 (MsrB3) induces cancer cell apoptosis through the activation of the intrinsic mitochondrial pathway. However, the mechanism through which MsrB3 deficiency results in cancer cell death is poorly understood. In this study, we investigated whether p53 and endoplasmic reticulum (ER) stress are involved in MsrB3 deficiency-induced cancer cell apoptosis using breast and colon cancer cells. MsrB3 depletion resulted in p53 down-regulation at the post-transcriptional level. MsrB3 deficiency induced cell death to a similar extent in both p53 wild-type (p53+/+) and null (p53−/−) cancer cells, suggesting that MsrB3 deficiency-induced apoptosis occurs independently of p53. MsrB3 deficiency significantly increased ER stress, which resulted in apoptosis. In addition, MsrB3 depletion activated the pro-apoptotic Bim molecule, which is essential for ER stress-induced apoptosis. MsrB3 deficiency increased cytosolic calcium levels, suggesting that MsrB3 down-regulation leads to a disturbance of calcium homeostasis in the ER, which consequently triggers ER stress. MsrB3 overexpression in MsrB3-depleted cells reduced ER stress, and was accompanied by at least partial recovery of cell viability. Taken together, our results suggest that MsrB3 plays a critical role in cancer cell apoptosis through the modulation of ER stress status.

Proteasome inhibitor MG132 induces thyroid cancer cell apoptosis by modulating the activity of transcription factor FOXO3a.

Proteasome inhibitors are promising antitumor drugs with preferable cytotoxicity in malignant cells and have exhibited clinical efficiency in several hematologic malignancies. P53-dependent apoptosis has been reported to be a major mechanism underlying. However, apoptosis can also be found in cancer cells with mutant-type p53, suggesting the involvement of p53-independent mechanism. Tumor suppressor forkhead Box O3 is another substrate of proteasomal degradation, which also functions partially through inducing apoptosis. The aim of this study was to explore the effect of proteasome inhibition on the expression and activity of forkhead Box O3 in thyroid cancer cells. Using flow cytometry, western blot, immunofluorescence staining and quantitative RT-PCR assays, we assessed proteasome inhibitor MG132-induced apoptosis in thyroid cancer cells and its effect on the expression and activity of forkhead Box O3. The resulted showed that MG132 induced significant apoptosis, and caused the accumulation of p53 protein in both p53 wild-type and mutant-type thyroid cancer cell lines, whereas the proapoptotic targets of p53 were transcriptionally upregulated only in the p53 wild-type cells. Strikingly, upon MG132 administration, the accumulation and nuclear translocation of transcription factor forkhead Box O3 as well as transcriptional upregulation of its proapoptotic target genes were found in thyroid cancer cells regardless of p53 status. Cell apoptosis was enhanced by ectopic overexpression while attenuated by silencing of forkhead Box O3. Altogether, we demonstrated that proteasome inhibitor MG132 induces thyroid cancer cell apoptosis at least partially through modulating forkhead Box O3 activity.

Abstract P3-07-07: Inhibition of death-associated protein kinase 1 enhances chemotherapy action against triple-negative breast cancer

Abstract Background: Triple negative breast cancers (TNBCs) are the most aggressive ER negative breast cancers with limited therapy strategies and poor prognosis. P53 gene is frequently mutated in approximately 80% of TNBCs. To identify novel molecular targets for ER negative breast cancer, particularly the more aggressive TNBC, we conducted a human kinome screen and identified death-associated protein kinase 1 (DAPK1) as one of the kinases that are highly expressed in ER negative breast cancer. Deletion or inhibition of DAPK1 suppresses growth of p53-mutant but not p53-wildtype breast cancer cells. Here we investigate whether DAPK1 inhibition will enhance chemotherapy action against p53-mutant TNBCs. Experimental design and methods: We performed experiments to test cell growth of p53-mutant TNBCs that were treated with DAPK1 siRNA or DAPK1 inhibitors in combination with different doses of chemotherapy drugs including 5-FU (5-Fluorouracil), doxorubicin, cisplatin, PARP inhibitor (BMN673), paclitaxel, gemcitabine and vinorelbine. Results: Our results show that DAPK1 inhibitors enhance the growth inhibitory effects of cisplatin and PARP inhibitor in p53-mutant TNBCs. Furthermore, combined DAPK1 inhibition (via siRNA knockdown) with cisplatin synergistically inhibits cell growth of p53-mutant TNBCs. Conclusion: DAPK1 is a novel, promising target for the treatment of triple-negative p53-mutant breast cancer. Our studies demonstrate that DAPK1 inhibition sensitizes TNBCs to the cytotoxic effects of cisplatin or the PARP inhibitor. We are now conducting studies to determine whether DAPK1 inhibition will sensitize TNBC tumors and patient-derived TNBC xenografts to the effects of cisplatin and PARP inhibition. These studies suggest that the combination of DAPK1 inhibition with drugs that interfere with DNA repair will be useful for the treatment of the most aggressive form of breast cancer, triple-negative breast cancer. Funding: This study was funded by a Susan G. Komen Promise grant (SAB12-00006 to P.H. Brown), a MD Anderson Knowledge Gap Moonshot grant (to P.H. Brown) and a Breast Cancer Research Foundation grant (BCRF 15101807, 2015–2016 to P.H. Brown). Citation Format: Zhao D, Zhao J, Mazumadar A, Bollu L, Shepherd J, Ma Y, Zhang Y, Hill JL, Savage MI, Brown PH. Inhibition of death-associated protein kinase 1 enhances chemotherapy action against triple-negative breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-07-07.

Raphanus sativus Sprout Causes Selective Cytotoxic Effect on p53-Deficient Human Lung Cancer Cells in vitro

Raphanus sativus L. (Brassicaceae) is widely consumed in many different forms worldwide. Its sprouts, in particular, are commonly consumed as a health food. R. stivus sprout has recently been shown to have anti-tumor activity on human colon cancer cells, suggesting that it may have potential use in.cancer prevention and treatment. The extent of this anti-tumor activity and its underlying mechanisms, however, remain to be investigated in other types-of cancer cells. In this study, we showed that the MeOH extract from R. sativus sprout exhibits significant but variable cytotoxic effects on human lung adenocarcinoma cells depending on their p53 status. The MeOH extract decreased the viability of p53-deleted human lung cancer cells (H1299 and Calu-6) by inducing apoptosis; this effect, however, did not occur for wild-type p53 cancer cells (A549), for cells expressing a p53 mutant lacking the C terminus (H1264), or for . non-tumor fibroblast cells (NIH3T3). Phytochemical analyses of the MeOH extract allowed us to identify and isolate 0-sitosterol as a major component of the MeOH extract. Direct treatment with P-sitosterol significantly reduced the viability of Calu-6 cells, suggesting that it may, in part, contribute to R. sativus sprout's anti-tumor activity. This work provides experimental evidence for a novel biological application of R. sativus sprout in treating human lung cancer, and it identifies the main component involved in this effect, further supporting its potential use as a functional food for cancer management.

TRIAD1 Is a Novel Transcriptional Target of p53 and Regulates Nutlin-3a-Induced Cell Death.

Nutlin-3a is a non-genotoxic, p53-activating, MDM2 inhibitor being investigated as an anticancer agent. Although Nutlin-3a selectively antagonizes the ubiquitin E3 ligase activity of MDM2, its efficacy is not entirely regulated by MDM2 levels in cancer cells. Here, we report that the cytotoxic effects of Nutlin-3a are regulated by TRIAD1 via a positive feedback loop with p53. We found that Nutlin-3a enhanced TRIAD1 transcription in a p53-dependent manner. Using in silico analysis and promoter luciferase assays, we demonstrated that p53-mediated transcription of TRIAD1 is mediated by a p53 consensus sequence in the TRIAD1 promoter region. Silencing TRIAD1 expression in wild-type p53 (p53WT ) cancer cells suppressed Nutlin-3a-mediated p53 activation and p53 target gene expression. These effects were enhanced in TRIAD1-overexpressing p53WT cancer cells, but not in p53-deficient cancer cells. Furthermore, TRIAD1 knockdown significantly reduced the growth inhibitory and cytotoxic effects of Nutlin-3a in p53WT cancer cells, as demonstrated by cell viability assays, cell cycle analysis, clonogenic growth, and soft-agar colony forming assays. Together, these data indicate that TRIAD1 regulates Nutlin-3a-mediated p53 activation and the cytotoxic activity of Nutlin-3a. J. Cell. Biochem. 118: 1733-1740, 2017. © 2016 Wiley Periodicals, Inc.

SHARPIN Facilitates p53 Degradation in Breast Cancer Cells

The ubiquitin binding protein SHAPRIN is highly expressed in human breast cancer, one of the most frequent female malignancies worldwide. Here, we perform SHARPIN depletion in breast cancer cells together with RNA sequencing. The global expression profiling showed p53 signaling as a potential SHARPIN target. SHARPIN depletion decreased cell proliferation, which effect could be rescue by p53 knocking down. Depletion SHARPIN significantly increases p53 protein level and its target genes in multiple breast cancer cell lines. Further experiment revealed that SHARPIN could facilitate p53 poly-ubiquitination and degradation in MDM2 dependent manner. Immuno-precipitation assay showed that SHARPIN associated with MDM2 and prolonged MDM2 protein stability. Analysis of public available database showed SHARPIN correlated with poor prognosis specifically in p53 wild-type breast cancer patients. Together, our finding revealed a novel modifier for p53/MDM2 complex and suggested SHARPIN as a promising target to restore p53 function in breast cancer.

The IGF-1R/AKT pathway determines cell fate in response to p53.

p53 that is activated in response to DNA-damaging stress can induce apoptosis or either transient or permanent cell cycle arrests. Apoptosis and permanent cell cycle arrest (senescence) are bona-fide tumor suppressor mechanisms through which p53 inhibits cancer cell survival. In contrast, transient cell cycle arrests induced by p53 can increase survival by allowing cells time to repair their DNA before proceeding with cell division. Mechanisms that control the choice of response to p53 (apoptosis vs arrest) are not fully understood. There is abundant crosstalk between p53 and the IGF-1R/AKT/mTORC1 signaling pathway. Recent studies indicate this crosstalk can determine the choice of response to p53. These findings have clear implications for the potential use of IGF-1R pathway inhibitors against p53 wild-type or p53-null or mutant cancers.

FBXW7 is involved in the acquisition of the malignant phenotype in epithelial ovarian tumors.

FBXW7 is a ubiquitin ligase that mediates ubiquitylation of oncoproteins, such as c‐Myc, cyclin E, Notch and c‐Jun. FBXW7 is a known tumor‐suppressor gene, and mutations in FBXW7 have been reported in various human malignancies. In this study, we examined the sequences of the FBXW7 and p53 genes in 57 ovarian cancer clinical samples. Interestingly, we found no FBXW7 mutations associated with amino acid changes. We also investigated FBXW7 expression levels in 126 epithelial ovarian tumors. FBXW7 expression was negatively correlated with the malignant potential of ovarian tumors. That is to say, FBXW7 expression levels in ovarian cancer samples were significantly lower than those in borderline and benign tumors (P < 0.01). FBXW7 expression levels in serous carcinoma samples were the lowest among four major histological subtypes. In addition, p53‐mutated ovarian cancer samples showed significantly lower levels of FBXW7 expression compared with p53 wild‐type cancer samples (P < 0.001). DNA methylation arrays and bisulfite PCR sequencing experiments revealed that 5′‐upstream regions of FBXW7 gene in p53‐mutated samples were significantly higher methylated compared with those in p53 wild‐type samples (P < 0.01). This data indicates that p53 mutations might suppress FBXW7 expression through DNA hypermethylation of FBXW7 5′‐upstream regions. Thus, FBXW7 expression was downregulated in ovarian cancers, and was associated with p53 mutations and the DNA methylation status of the 5′‐upstream regions of FBXW7.

Abstract LB-016: Nutlin and oxaliplatin induce p53-dependent addiction to FLIP

Abstract Background: The tumour suppressive functions of the p53 transcription factor are inactivated through mutations or suppressed through non-mutational mechanisms in almost all cancer cells. This work focusses on identifying mechanisms through which, tumours retaining wild-type p53 evade apoptosis in response to chemotherapies and p53 agonists such as Nutlin-3A. Methods: A panel of matched p53 wild-type and deficient colorectal cancer cell line models were studied, using Nutlin-3A and oxaliplatin as direct and indirect p53 activating agents. A number of molecular (Western blots, RT-PCR), phenotypic (cell death) and genomic analyses were used to investigate the importance of p53 and its downstream transcriptional programs. Results: We demonstrate the importance of p53 in priming cancer cell sensitivity to apoptotic cell death following treatment with Nutlin-3A or oxaliplatin through activation of pro-apoptotic p53 targets, such as TRAIL-R2, Fas/CD95, PUMA and NOXA. Surprisingly, our analysis further reveals that this priming is accompanied by a parallel early p53-dependent pro-survival response that imposes a critical dependence on the caspase-8 inhibitor FLIP. Preventing FLIP up-regulation with siRNA resulted in synergistic increases in apoptosis in response to Nutlin-3A and oxaliplatin in p53 wild-type, but not p53 null cells. p53-mediated FLIP up-regulation was blocked by pharmacological inhibition of HDACs1-3 using Entinostat, which caused hyper-acetylation of the p53 C-terminus and altered transactivation of a number of other p53 apoptotic target genes. Notably, the synergy between Nutlin-3A or oxaliplatin and Entinostat was abrogated in cells over-expressing FLIP trans-genes or in cells in which caspase-8 was depleted using RNAi. Conclusion: These results uncover a p53-regulated apoptosis priming event, in which a number of key pro-apoptotic genes are upregulated in response to direct p53 agonism (Nutlin-3A) or indirect activation downstream of DNA damage (oxaliplatin). However, immediate commitment to apoptosis is prevented by the concomitant p53-mediated up-regulation of FLIP. This may be a mechanism that has evolved to allow DNA damage repair time to take place, while priming the cell to undergo apoptosis if the damage is irreparable. From a cancer therapeutics point-of-view, this work identifies FLIP upregulation in p53 wild-type cancers as a key target for overcoming resistance to direct and indirect p53 activating therapies. Citation Format: Alexander McIntyre, Fiammetta Falcone, Patrick G. Johnston, Daniel B. Longley, Simon S. McDade. Nutlin and oxaliplatin induce p53-dependent addiction to FLIP. [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 LB-016.

Abstract 3512: Cyclopamine enhances TRAIL-induced apoptosis by induction of DR5 via ER stress in gastric cancer cells

Abstract Activation of sonic hedgehog (Shh) signaling has been involved in progression of various cancers. Cyclopamine uses as effective treatment for cancers in which hedgehog signaling is overexpressed. In this study, we elucidated that cyclopamine sensitizes to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in gastric cancer cells. Single treatment with cyclopamine or TRAIL could not induce significant cytotoxicity in gastric cancer cells. However, combination treatment of TRAIL with cyclopamine effectively led to caspase dependent apoptosis. We found that cyclopamine increased ER (Endoplasmic Reticulum) stress level in gastric cancer cells, and using ER stress inhibitor attenuated the cell death by cyclopamine. As further underlying mechanism, induction of ER stress by cyclopamine caused upregulation of JNK (c-Jun N-terminal kinases) protein, and then accumulation of p53 protein. We conducted experiment using p53 wild-type and p53-mutated gastric cancer cells, and this of particular importance since p53 wild-type gastric cancer cells had more significant efficacy than p53-mutated cells by cyclopamine. Accumulation of p53 increased DR5 protein. Taken together, we showed that cyclopamine sensitizes to TRAIL-induced apoptosis in gastric cancer cells. Citation Format: Yoo Jin Na, Dae-Hee Lee, Jung Lim Kim, Bo Ram Kim, Seong Hye Park, Byung-Wook Min, Sun Il Lee, SangHee Kang, Sung Yup Joung, Suk Young Lee, Hong-Jun Kim, Sang Cheul Oh. Cyclopamine enhances TRAIL-induced apoptosis by induction of DR5 via ER stress in gastric cancer cells. [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 3512.

Abstract 4855: Discovery of NVP-HDM201 - First disclosure of a Next-Generation Mdm2 inhibitor with superior characteristics

Abstract Activation of p53 by blocking the p53-Mdm2 interaction using non-peptidic small-molecule inhibitors has been pursued for many years as a promising cancer therapeutic strategy. We disclose the identity of NVP-HDM201, a novel, highly optimized and selective inhibitor of the p53-Mdm2 interaction. NVP-HDM201 binds to human Mdm2 protein with a sub-nanomolar Ki value, activates p53 and induces robust p53-dependent cell cycle arrest and apoptosis in human p53 wild-type tumor cells. The activity and selectivity of NVP-HDM201 have been tested and confirmed across a panel of cancer cell lines and the molecule displays desirable pharmacokinetic and pharmacodynamic profiles in animals together with excellent oral bioavailability. Application of NVP-HDM201 using various dosing schedules triggers rapid and sustained activation of p53-dependent pharmacodynamic biomarkers resulting in tumor regression in multiple xenografted models of p53 wild-type human cancers. We report here how a promising lead series was discovered and how innovative medicinal chemistry efforts led to further optimization of the potency and physico-chemical properties, culminating in the discovery of NVP-HDM201. The superior characteristics of the compound allowed the fast progression of the compound into the clinic where NVP-HDM201 is currently in Phase 1 clinical trials both as a single agent and as a combination partner in patients pre-selected for p53 wild-type tumors. Citation Format: Philipp Holzer, Patrick Chène, Stéphane Ferretti, Pascal Furet, Tobias Gabriel, Bjoern Gruenenfelder, Vito Guagnano, Francesco Hofmann, Joerg Kallen, Robert Mah, Keiichi Masuya, Rita Ramos, Stephan Ruetz, Caroline Rynn, Thérèse Stachyra-Valat, Stefan Stutz, Andrea Vaupel, Sébastien Jeay. Discovery of NVP-HDM201 - First disclosure of a Next-Generation Mdm2 inhibitor with superior characteristics. [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 4855.

5-Caffeoylquinic acid inhibits invasion of non-small cell lung cancer cells through the inactivation of p70S6K and Akt activity: Involvement of p53 in differential regulation of signaling pathways.

In the present study, we investigated the effects and molecular mechanism of 5-caffeoylquinic acid (5-CQA), a natural phenolic compound isolated from Ligularia fischeri, on cell invasion, proliferation and adhesion in p53 wild-type A549 and p53-deficient H1299 non-small cell lung cancer (NSCLC) cells. 5-CQA abrogated mitogen-stimulated invasion, but not proliferation, in both A549 and H1299 cells. In addition, 5-CQA inhibited mitogen-stimulated adhesion in A549 cells only. Anti-invasive activity of 5-CQA in A549 cells was mediated by the inactivation of p70(S6K)-dependent signaling pathway. In contrast, in H1299 cells the inactivation of Akt was found to be involved in 5-CQA-mediated inhibition of cell invasion. Collectively, these findings demonstrate the pharmacological roles and molecular targets of 5-CQA in regulating NSCLC cell fate, and suggest further evaluation and development of 5-CQA as a potential therapeutic agent for the treatment and prevention of lung cancer.

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.

Clinical Overview of MDM2/X-Targeted Therapies.

MDM2 and MDMX are the primary negative regulators of p53, which under normal conditions maintain low intracellular levels of p53 by targeting it to the proteasome for rapid degradation and inhibiting its transcriptional activity. Both MDM2 and MDMX function as powerful oncogenes and are commonly over-expressed in some cancers, including sarcoma (~20%) and breast cancer (~15%). In contrast to tumors that are p53 mutant, whereby the current therapeutic strategy restores the normal active conformation of p53, MDM2 and MDMX represent logical therapeutic targets in cancer for increasing wild-type (WT) p53 expression and activities. Recent preclinical studies suggest that there may also be situations that MDM2/X inhibitors could be used in p53 mutant tumors. Since the discovery of nutlin-3a, the first in a class of small molecule MDM2 inhibitors that binds to the hydrophobic cleft in the N-terminus of MDM2, preventing its association with p53, there is now an extensive list of related compounds. In addition, a new class of stapled peptides that can target both MDM2 and MDMX have also been developed. Importantly, preclinical modeling, which has demonstrated effective in vitro and in vivo killing of WT p53 cancer cells, has now been translated into early clinical trials allowing better assessment of their biological effects and toxicities in patients. In this overview, we will review the current MDM2- and MDMX-targeted therapies in development, focusing particularly on compounds that have entered into early phase clinical trials. We will highlight the challenges pertaining to predictive biomarkers for and toxicities associated with these compounds, as well as identify potential combinatorial strategies to enhance its anti-cancer efficacy.

Abstract A01: A comparison of the mechanisms and cytotoxic activity of dianhydrogalactitol (VAL-083) to cisplatin in ovarian tumor models harboring wild-type and mutant p53.

Abstract Ovarian cancers are usually treated with platinum-based therapies, which produce a 70% response rate. However, many patients relapse as tumors become resistant to cisplatin and carboplatin, resulting in a 5-year survival rate of about 20%. Onset of drug resistance is a major factor limiting the clinical utility of platinum-based therapeutic regimens drugs and, therefore, new agents are needed to circumvent resistance. Dianhydrogalactitol (VAL-083) is a bi-functional alkylating agent, whose cytotoxic activity is due to the formation of DNA cross links at the N7 position of guanine. Unlike cisplatin and carboplatin, which predominantly form intrastrand DNA cross-links, VAL-083 derives its anti-cancer activity interstrand DNA cross-links. More importantly, VAL-083 has demonstrated clinical activity against a range of tumor types, including ovarian cancer in historical NCI-sponsored clinical studies. Platinum drug resistance is normally ascribed to several mechanisms, with mutation in wild-type p53 playing a critical role, particularly in high-grade ovarian serous carcinoma (HGOSC), where the incidence of this mutation can be substantial. However, recent analysis of the TCGA database indicates that the survival rate in wild-type p53 HGOSC is no better or perhaps even worse. Using a 5-day MTT assay and fitting a sigmoidal curve to the dose-response data to determine IC50 values, we have similarly found that resistance to cisplatin in a panel of ovarian tumor models is greater when p53 is wild-type (median IC50: 4-7 µM vs. 1-2 µM for mutant/null p53 models; in comparison, sensitive wild-type p53 A2780 cells have an IC50 of 0.2-0.3 μM). Previous studies in our lab suggest that factors downstream from p53, including MDM4 and p21, may contribute to cisplatin-resistance in ovarian cancer models with high cisplatin-resistance and wild-type 53, like 2780CP-16. We thus sought to investigate the potential of VAL-083 to circumvent cisplatin-resistance in five p53 wild-type ovarian cancer models: one cisplatin-sensitive A2780, and four cisplatin-resistant 2780CP-16, OVCAR-10, Hey and OVCA-433. IC50 values of VAL-083 were generated using the MTT assay and sigmoidal curve fitting of data, as described above. The baseline IC50 for VAL-083 against A2780 cell was about 0.5 μM. The IC50 for VAL-083 in the cisplatin-resistant cell-lines 2780CP-16, OVCAR-10, Hey and OVCA-433 were 4- to 7-fold greater; however, VAL-083 was substantially more potent in comparison to cisplatin in these models where corresponding IC50 values were 10- to over 25-fold greater. These results demonstrate that there is only partial cross-resistance between cisplatin and VAL-083 further suggesting distinct modes of action for the two drugs. In order to examine whether the partial circumvention of cisplatin-resistance was p53-dependent, cytotoxicity was determined in isogenic HCT-116p53-/- and HCT-116p53+/+ models. These studies demonstrated that loss of p53 increased resistance to cisplatin by 2-5-fold whereas loss of p53 only increased resistance to VAL-083 about 1.7-fold. These results suggest that VAL-083 is less dependent on p53 for its cytotoxic activity. Immunoblots confirmed this in 2780CP-16 cells, where VAL-083 was more effective than cisplatin at increasing p53 and p21 levels, and induced relatively greater Ser-15 and Ser-20 phosphorylations, further supporting different modes of action for the two drugs. In contrast, both drugs were equally effective in inducing these markers of DNA damage in A2780 cells. The non-overlapping mechanisms of action suggested a potential therapeutic benefit for combinations of VAL-083 with cisplatin. We have previously reported that the combination of VAL-083 with cisplatin in wild-type p53 NSCLC models H460 and A549, and mutant p53 NSCLC H1975 demonstrated significant super-additivity (p&amp;lt;0.05) and synergy (CI &amp;lt; 1) in all three cell-lines. Taken together these results demonstrate the effectiveness of VAL-083 against refractory cisplatin-resistant ovarian cancers and raise the potential for treatment of platinum-resistant ovarian cancers or a combination regimen with cisplatin. (Supported in part by NCI RO1 CA160687 to ZHS). Citation Format: Jeffrey A. Bacha, Michelle Martinez-Rivera, Guanghan He, Xiaolei Xie, Anne Steino, Sarath Kanekal, Dennis M. Brown, Zahid H. Siddik. A comparison of the mechanisms and cytotoxic activity of dianhydrogalactitol (VAL-083) to cisplatin in ovarian tumor models harboring wild-type and mutant p53. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A01.

Upregulation of BTG1 enhances the radiation sensitivity of human breast cancer in vitro and in vivo.

X-ray-based radiotherapy is one of the most effective therapeutic strategies for breast cancer patients. However, radioresistance and side-effects continue to be the most challenging issues. B-cell translocation gene 1 (BTG1) is a member of the BTG/Tob family, which inhibits cancer growth and promotes apoptosis. We, therefore, hypothesized that BTG1 plays an important role in the radiosensitivity of breast cancer cells. In the present study, breast cancer cell lines that stably overexpressed BTG1 were used to investigate the effects of BTG1 on cell radiosensitivity in vitro. We found that overexpression of BTG1 enhanced the radiosensitivity both of p53-mutant breast cancer MDA-MB-231 cells and p53 wild-type breast cancer MCF-7 cells. We also found that overexpression of BTG1 along with irradiation induced cell cycle G2/M phase arrest, promoted the formation of reactive oxygen species (ROS), increased the rate of chromosomal aberrations and increased cell apoptosis. Further investigation indicated that BTG1 overexpression along with irradiation was involved in inhibition of the PI3K/Akt signaling pathway. Importantly, the finding that BTG1 promoted ionizing radiosensitivity of breast cancer cells in vitro was confirmed in an animal model. Taken together, our data suggest that BTG1 overexpression combined with radiation therapy increases the therapeutic efficacy of breast cancer treatment via regulation of the cell cycle and apoptosis-related signaling pathways.

Abstract 2125: Cis-mediated regulation of mRNA translation initiation of p53 family members

Abstract The p53 family of transcription factors comprises several proteins produced by three genes, TP63, TP63 and TP73, through alternative promoters and splicing, that exhibit critical functions for cell homeostasis and act as prominent tumor suppressors. The p53 pathway is often activated in stress conditions during which the global cap-dependent translation is inhibited. Although p53 protein is highly regulated at post-translational level, cells maintain continuous p53 protein synthesis also taking advantage of the presence of two Internal Ribosome Entry Sites (IRESs) elements residing within the p53 mRNA. The first IRES is located in the 5′UTR of the full-length isoform, the second is located into the protein-coding region and mediates the translation of a ΔN-p53 isoform. IRES elements are complex RNA structural elements in 5′UTRs that can mediate cap-independent initiation of translation under stress conditions. Even though an IRES activity has been already established for p53, no studies are available for p63 and p73 transcripts. This work is mainly focused on the regulation of translation initiation efficiency for the p53 family members p63 and p73. For this purpose, we have cloned the 5′UTR from human full-length p53 (as positive control), TA-p63, TA*-p63, TA-p73 and ΔN-p63 and ΔN-p73 transcript isoforms in a bicistronic reporter construct (named pRuF, where R stands for Renilla reniformis luciferase cDNA, u for 5′UTR tested and F for Firefly luciferase cDNA). The c-Myc and p16 or β-globin and β-actin 5′UTRs were included as positive or negative controls. We performed gene reporter assays in MCF7 and HCT116 p53 wild-type cancer cell lines and in their derivative clones with reduced (shp53) or null (p53-/-) p53 expression. It has been previously demonstrated that p53, through transcriptional repression of fibrillarin (FBL) is able to negatively impact on rRNA methylation pattern and IRES-dependent translation, hence p53 status can be an important factor to unmask cap-independent translation initiation. Results showed that TA*-p63, TA-p73 and ΔN-p73 can potentially contain an IRES-like sequence in their 5′UTR. Translation stimulation was increased in MCF7 cells with p53 was knocked-down. Treatment with the mTOR inhibitors Rapamycin and Torin1, that inhibit cap-dependent translation, led to an enhanced relative activity of the putative IRES contained in TA-p73. Interestingly, differences in responses of the various 5′UTRs between the two cell lines were observed. Based on these initial observations, the identification of different and tissue specific RNA binding proteins targeting the distinct p63 and p73 5′UTRs will be pursued with the aim to dissect a mechanism regulating mRNA translation initiation that could play an important role in development as well as in cancer progression. Note: This abstract was not presented at the meeting. Citation Format: Alessandra Bisio, Alberto Inga. Cis-mediated regulation of mRNA translation initiation of p53 family members. [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 2125. doi:10.1158/1538-7445.AM2015-2125

Abstract 1992: Evidence for modulation of FoxM1 by p21 in ovarian cancer

Abstract The oncogenic transcription factor forkhead box M1 (FoxM1) is overexpressed in many cancers, including 84% of ovarian cancer and plays a role in DNA repair, mitotic checkpoint, cell proliferation, and cancer drug resistance. Similar to Her2 in breast cancer, the constitutive expression of FoxM1 makes it a plausible gene target for novel anti-cancer therapies, but the regulation of FoxM1 has yet to be elucidated. Evidence suggests FoxM1 up-regulation is a result of TP53 mutations, however, no TP53 response element has been found within the FoxM1 promoter, thus there is likely another molecule mediating p53-induced FoxM1 overexpression. We hypothesize that cyclin-dependent kinase inhibitor 1a, p21, is involved in the regulation of FoxM1 by p53 in ovarian cancer. To test this hypothesis, we knocked down p21 expression with siRNA and shRNA in two wild-type p53 cancer cell lines, NCI-H23 and A2780. Knock down of p21 resulted in a mirrored increase in FoxM1 protein level in both cell lines as detected by Western blot. The addition of nutlin-3 (10 μM), a p53 stabilizer, to these p21 knocked down cell lines restored some p21 expression 24 hour post-treatment, which led to a subsequent decrease in FoxM1 levels compared to the corresponding DMSO-treated p21 knock down. These results suggest p21 is a negative regulator of FoxM1 in cancer cell lines. To further investigate this pathway, the same two cancer cell lines were treated with the FoxM1 inhibitor thiostrepton (10 μM) and protein levels were measured over a time course of 48 hours. FoxM1 protein levels were reduced following 8 hours, a time point where p21 protein levels peaked. This finding was further support by qRT-PCR analysis of A2780 ovarian cancer cell mRNA collected 0, 6, 8, 9, 10, 11, and 12 hours following thiostrepton (10 μM) treatment. In this experiment, p21 mRNA levels peaked 6 hours post-treatment, with FoxM1 mRNA levels reduced at 8 hours post-treatment, relative to control. Although thiostrepton has been shown to directly bind and inhibit the transcription of FoxM1, these results suggest that thiostrepton may also induce p21, potentiating FoxM1 inhibition. Thiostrepton has previously been shown to induce cancer cell death and reduce tumor burden, therefore further supporting FoxM1 as a potential drug target as well as encouraging the need to understand regulators involved in this pathway. Citation Format: Jill Madden, Jeremy Chien. Evidence for modulation of FoxM1 by p21 in ovarian cancer. [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 1992. doi:10.1158/1538-7445.AM2015-1992

Direct relationship between the level of p53 stabilization induced by rRNA synthesis-inhibiting drugs and the cell ribosome biogenesis rate.

Many drugs currently used in chemotherapy work by hindering the process of ribosome biogenesis. In tumors with functional p53, the inhibition of ribosome biogenesis may contribute to the efficacy of this treatment by inducing p53 stabilization. As the level of stabilized p53 is critical for the induction of cytotoxic effects, it seems useful to highlight those cancer cell characteristics that can predict the degree of p53 stabilization following the treatment with inhibitors of ribosome biogenesis. In the present study we exposed a series of p53 wild-type human cancer cell lines to drugs such as actinomycin D (ActD), doxorubicin, 5-fluorouracil and CX-5461, which hinder ribosomal RNA (rRNA) synthesis. We found that the amount of stabilized p53 was directly related to the level of ribosome biogenesis in cells before the drug treatment. This was due to different levels of inactivation of the ribosomal proteins-MDM2 pathway of p53 digestion. Inhibition of rRNA synthesis always caused cell cycle arrest, independent of the ribosome biogenesis rate of the cells, whereas apoptosis occurred only in cells with a high rDNA transcription rate. The level of p53 stabilization induced by drugs acting in different ways from the inhibition of ribosome biogenesis, such as hydroxyurea (HU) and nutlin-3, was independent of the level of ribosome biogenesis in cells and always lower than that occurring after the inhibition of rRNA synthesis. Interestingly, in cells with a low ribosome biogenesis rate, the combined treatment with ActD and HU exerted an additive effect on p53 stabilization. These results indicated that (i) drugs inhibiting ribosome biogenesis may be highly effective in p53 wild-type cancers with a high ribosome biogenesis rate, as they induce apoptotic cell death, and (ii) the combination of drugs capable of stabilizing p53 through different mechanisms may be useful for treating cancers with a low ribosome biogenesis rate.

Abstract P6-05-02: Expression of ING1b-derived peptide inhibits viability of breast cancer cells and promotes apoptosis

Abstract The ING1b protein is a type II tumor suppressor and a stoichiometric member of HDAC-containing protein complexes. Primarily by altering chromatin structure, ING1b contributes to regulation of gene expression, senescence and apoptosis. Mislocalization and decreased levels of ING1b are commonly observed in human tumors and cancer cell lines. Multiple independent studies in varied cell types report that ING1b overexpression promotes apoptosis. Since the inactivation of apoptosis pathways is frequent in cancer cells, modulating ING1b expression in tumors may serve as a viable approach for cancer therapy. We have defined the minimal ING1b region that confers apoptotic function as estimated by the levels of PARP cleavage, FACS analysis and using an Annexin V binding assay. We have established that ING1b-derived peptides containing its third alpha helix (A3H) and NLS/NTS domains are able to induce apoptosis at levels comparable to those of full length ING1b. The A3H-NLS/NTS peptide exhibited strong nucleolar localization characteristic of full length ING1b. Cells overexpressing the full length ING1b and A3H-NLS/NTS peptide showed similar changes in cell morphology characteristic of apoptosis and exhibited increased levels of PARP cleavage. While the A3H region that includes the N-terminal part of the highly conserved Lamin Interacting Domain (LID) was necessary but not sufficient, the NLS/NTS domain that mediates nuclear and nucleolar localization of ING1b was required and partially sufficient for induction of apoptosis. Adenoviral-mediated expression of A3H-NLS/NTS peptide in cells of osteosarcoma, glioblastoma and breast cancer origins resulted in strong reduction of cell viability. Depending on the cell line, treating cells with 45 MOI of virus expressing A3H-NLS/NTS peptide resulted in a 60 - 85% decrease in cancer cell survival compared to cells treated with the control construct (Ad-GFP), and the highly transformed triple negative tumorigenic MDA-MB-468 breast cancer line was sensitive to the peptide when infected with 5 MOI of A3H-NLS/NTS adenovirus. These pro-apoptotic effects were found to be dose and time dependent. Furthermore, using p53 wild-type, p53 mutant and p53-null cancer cell lines we demonstrated that the effects of A3H-NLS/NTS expression on cell survival and apoptosis were independent of p53-status. The evaluation of the synergy between the A3H-NLS/NTS peptide and common chemotherapeutic agents is currently ongoing. Our long-term goal is to develop ING1b-based therapeutics that can be used as an adjuvant therapy in combination with the existing breast cancer treatments. Citation Format: Oleksandr Boyko, Karl Riabowol. Expression of ING1b-derived peptide inhibits viability of breast cancer cells and promotes apoptosis [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-05-02.