Overexpression Of Wild-type P53 Research Articles

Modulation of p53 expression in cancer-associated fibroblasts prevents peritoneal metastasis of gastric cancer

Cancer-associated fibroblasts (CAFs) in the tumor microenvironment are associated with the establishment and progression of peritoneal metastasis. This study investigated the efficacy of replicative oncolytic adenovirus-mediated p53 gene therapy (OBP-702) against CAFs and peritoneal metastasis of gastric cancer (GC). Higher CAF expression in the primary tumor was associated with poor prognosis of GC, and higher CAF expression was also observed with peritoneal metastasis in immunohistochemical analysis of clinical samples. And, we found transcriptional alteration of p53 in CAFs relative to normal gastric fibroblasts (NGFs). CAFs increased the secretion of cancer-promoting cytokines, including interleukin-6, and gained resistance to chemotherapy relative to NGFs. OBP-702 showed cytotoxicity to both GC cells and CAFs but not to NGFs. Overexpression of wild-type p53 by OBP-702 infection caused apoptosis and autophagy of CAFs and decreased the secretion of cancer-promoting cytokines by CAFs. Combination therapy using intraperitoneal administration of OBP-702 and paclitaxel synergistically inhibited the tumor growth of peritoneal metastases and decreased CAFs in peritoneal metastases. OBP-702, a replicative oncolytic adenovirus-mediated p53 gene therapy, offers a promising biological therapeutic strategy for peritoneal metastasis, modulating CAFs in addition to achieving tumor lysis.

P53 Promoted Ferroptosis in Ovarian Cancer Cells Treated with Human Serum Incubated-Superparamagnetic Iron Oxides.

MethodsIn this study, we used MTT assays to demonstrate that a combination of SPIO-Serum and wild-type p53 overexpression can reduce ovarian cancer cell viability in vitro. Prussian blue staining and iron assays were used to determine changes in intracellular iron concentration following SPIO-Serum treatment. TEM was used to evaluate any mitochondrial damage induced by SPIO-Serum treatment, and Western blot was used to evaluate the expression of the iron transporter and lipid peroxidation regulator proteins. JC-1 was used to measure mitochondrial membrane potential, and ROS levels were estimated by flow cytometry. Finally, xCT protein expression and mitochondrial ROS levels were confirmed using fluorescence microscopy.ResultsSPIO-Serum effectively induced lipid peroxidation and generated abundant toxic ROS. It also facilitated the downregulation of GPX4 and xCT, ultimately resulting in iron-dependent oxidative death. These effects could be reversed by iron chelator DFO and lipid peroxidation inhibitor Fer-1. SPIO-Serum treatment disrupted intracellular iron homeostasis by regulating iron uptake and the cells presented with missing mitochondrial cristae and ruptured outer mitochondrial membranes. Moreover, we were able to show that p53 contributed to SPIO-Serum-induced ferroptosis in ovarian cancer cells.ConclusionSPIO-Serum induced ferroptosis and overexpressed p53 contributed to ferroptosis in ovarian cancer cells. Our data provide a theoretical basis for ferroptosis as a novel cell death phenotype induced by nanomaterials.

Diagnosis and management of a solitary colorectal juvenile polyp in an adult during follow-up for ulcerative colitis: A case report

BACKGROUNDJuvenile polyps are the most common type of polyps in children but are rare in adults. Inflammatory bowel disease (IBD) patients have a similar spectrum of symptoms as patients with juvenile polyps. Both patients with juvenile polyps and those with active IBD have high fecal calprotectin levels. Four cases of children with ulcerative colitis (UC) with solitary juvenile polyps and one case of an adult with UC with juvenile polyposis syndrome have been reported upon diagnosis of UC, while there have been no cases of adults with UC with solitary juvenile polyp reported in the literature.CASE SUMMARYA 37-year-old man with a 12-year history of UC was admitted to our clinic because of increased stool frequency. UC was diagnosed at the age of 25. As the lesion was confined to the rectum, sulfasalazine suppositories or mesalazine suppositories were used. The patient was followed in an outpatient clinic, and colonoscopy was performed every one or two years. The latest examination was undertaken three years prior in the presence of proctitis. Recently, the patient complained of three to five bowel movements a day. There was mucus in the stool but no visible blood. Colonoscopy revealed a solitary polyp, about 1.5 cm in diameter, with a short and broad peduncle in the transverse colon surrounded by congestive and edematous mucosa. The patient had no family history of colorectal polyps or cancer. The polyp was successfully removed by endoscopic mucosal resection. Histopathological examination revealed that the polyp was a juvenile polyp without any malignant signs. Immunohistochemical staining for p53 showed wild-type expression and p53 overexpression was not detected. Ki-67 labeling index was 3%.CONCLUSIONThis is the first case of an adult UC patient with a solitary juvenile polyp at the 12-year follow-up. The correlation between juvenile polyps and the activity of IBD needs further study.

Mutant P53 induces MELK expression by release of wild-type P53-dependent suppression of FOXM1

Triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer, and is associated with a poor prognosis due to frequent distant metastasis and lack of effective targeted therapies. Previously, we identified maternal embryonic leucine zipper kinase (MELK) to be highly expressed in TNBCs as compared with ER-positive breast cancers. Here we determined the molecular mechanism by which MELK is overexpressed in TNBCs. Analysis of publicly available data sets revealed that MELK mRNA is elevated in p53-mutant breast cancers. Consistent with this observation, MELK protein levels are higher in p53-mutant vs. p53 wild-type breast cancer cells. Furthermore, inactivation of wild-type p53, by loss or mutation of the p53 gene, increases MELK expression, whereas overexpression of wild-type p53 in p53-null cells reduces MELK promoter activity and MELK expression. We further analyzed MELK expression in breast cancer data sets and compared that with known wild-type p53 target genes. This analysis revealed that MELK expression strongly correlates with genes known to be suppressed by wild-type p53. Promoter deletion studies identified a p53-responsive region within the MELK promoter that did not map to the p53 consensus response elements, but to a region containing a FOXM1-binding site. Consistent with this result, knockdown of FOXM1 reduced MELK expression in p53-mutant TNBC cells and expression of wild-type p53 reduced FOXM1 expression. ChIP assays demonstrated that expression of wild-type p53 reduces binding of E2F1 (a critical transcription factor controlling FOXM1 expression) to the FOXM1 promoter, thereby, reducing FOXM1 expression. These results show that wild-type p53 suppresses FOXM1 expression, and thus MELK expression, through indirect mechanisms. Overall, these studies demonstrate that wild-type p53 represses MELK expression by inhibiting E2F1A-dependent transcription of FOXM1 and that mutation-driven loss of wild-type p53, which frequently occurs in TNBCs, induces MELK expression by suppressing FOXM1 expression and activity in p53-mutant breast cancers.

Rare Case of Metastatic Ovarian Clear Cell Carcinoma With an In Vitro Fertilization Pregnancy

Abstract The risk of ovarian malignancy is rare in pregnancy with a reported incidence of approximately 1 in 50,000. Ovarian clear cell carcinoma (OCCC) accounts for 5% to 10% of all ovarian carcinomas with mean age of 50 to 55 years and is often associated with endometriosis. We present a case of 38-year-old G2P1 Asian female with an in vitro fertilization pregnancy and history of endometriosis who presented with the right adnexal mass. The transvaginal ultrasound performed during the early pregnancy was suggestive of endometrioma or desmoid tumor. At 32 weeks, the patient presented to the emergency department with preeclampsia and severe headache. An emergency cesarean section was performed due to worsening of the patient’s condition. An ovarian mass arising from the right ovary and with a friable and hemorrhagic internal surface was excised, which weighed 63 g and measured 14.0 × 10.5 × 3.0 cm. Its external surface was smooth, hemorrhagic, but without any excrescences. The serial sectioning revealed multiple cystic lesions filled with necrotic material overall occupying 80% of the mass. Microscopically, the viable ovarian tissue demonstrated tubulocystic/papillary architecture lined by hyperchromatic hobnail cells along with focal areas of solid sheets of tumor cells displaying clear cytoplasm. Immunohistochemical stains for Wilm’s tumor 1 and p53 were negative while positive for PAX8. These findings were indicative of OCCC without overexpression of wild-type p53. The PET scan performed few weeks after delivery showed widespread metastasis. We present a unique case of metastatic OCCC removed at the time of cesarean section in the late-preterm period. Our case not only emphasizes the need for definitive treatment option for endometriosis but also a close imaging surveillance of all masses diagnosed during pregnancy in women with risk factors such as endometriosis and Asian ethnicity. Furthermore, our study advocates the need for possible guidelines for management of such rare cases.

Abstract 3472: The role of mutant p53 R248W in oral squamous cell carcinoma

Abstract TP53 is the most commonly mutated gene in oral squamous cell carcinoma (OSCC). It is known that mutant p53 has not only loss of function but gain of function. Cytokeratin 17 (K17) has been shown to promote tumorigenesis and aggressiveness in OSCCs and other various carcinomas. Although p53 reportedly induces K17 transcription by irradiation, the molecular interaction between p53 and K17 in OSCCs remains unknown. To address this issue, we investigated the relationship between p53 and K17 using SAS cells with nonsense mutation and Ca9-22 cells with R248W mutation in the TP53 gene. Western blot and immunohistochemistry showed higher K17 expression in SAS cells and virtually no K17 expression in Ca9-22 cells, suggesting negative correlation between p53 and K17 expression. ChIP analysis revealed that p53 bound specifically to the tentative p53 binding site in the promoter region of the K17 gene. Unexpectedly, however, overexpression of wild-type (wt) p53 did not suppress K17 expression in SAS cells, whereas knock down of mutant (mt) p53 significantly upregulated K17 expression in Ca9-22 cells, raising a possibility that only R248W mtp53 suppresses K17 expression. Furthermore, overexpression of wtp53 and R248Q mtp53 did not suppressed K17 expression in Ca9-22 cells with knock down of p53, whereas R248W mtp53 overexpression clearly showed suppression of K17. Taken together, R248W mtp53 acts as a direct transcriptional repressor of K17 in OSCCs, reducing aggressiveness of the carcinoma cells. Citation Format: Mayu Enaka, Masako Nakanishi, Yasuteru Muragaki. The role of mutant p53 R248W in oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3472.

Abstract 1409: p53 negatively regulates keratin 17 expression in oral squamous cell carcinoma

Abstract Background: Cytokeratin 17 (K17) has been shown to promote tumorigenesis and aggressiveness in oral squamous cell carcinomas (OSCCs) and other various carcinomas. Although p53 reportedly induces K17 transcription by irradiation, the correlation between p53 and K17 in OSCCs remains unknown. Methods: Human OSCC cell lines SAS and Ca9-22 were examined by Western blot, immunohistochemistry, ChIP assays, and overexpression of wild-type p53. Biopsy specimens from patients with OSCC were used for immunohistochemistry.Results: Western blot and immunohistochemistry showed that K17 expression negatively correlated with p53 expression. In biopsy specimens, positive staining for K17 was observed in OSCC cells that were negative for p53 in a mutually exclusive manner. SAS cells, which have higher K17 expression and almost negative p53 expression, showed more aggressive characteristics than Ca9-22 cells. A mutation to cause a premature stop codon was identified in the TP53 gene in SAS cells by DNA sequencing, whereas no mutation in the TP53 gene was identified in Ca9-22 cells. In addition, ChIP analysis showed that p53 bound specifically to the promoter region of the K17 gene. Furthermore, overexpression of wild-type p53 suppressed K17 expression in SAS cells. Conclusion: p53 acts as a direct transcriptional repressor of K17 in OSCCs. The interaction between p53 and K17 may regulate aggressiveness of the carcinoma cells. Citation Format: Mayu Enaka, Masako Nakanishi, Yasuteru Muragaki. p53 negatively regulates keratin 17 expression in oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1409.

Abstract 2532: Wild-type p53 represses MELK expression by blocking the recruitment of FOXM1 to MELK promoter

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. To identify potential druggable targets, previously we performed a gene expression analysis and identified protein kinases that are highly expressed in TNBCs. In this study, we investigated the molecular mechanism through which wild-type p53 represses MELK expression in TNBC cells. Through these studies, we discovered that the expression of Maternal Embryonic Leucine zipper Kinase (MELK) is highly elevated in TNBC when wild-type p53 is lost or mutated. We also show that overexpression of wild-type p53 represses MELK expression without binding to MELK promoter. Experimental Design and Methods: To identify the p53-responsive region in MELK promoter, we have made multiple promoter deletions using PCR deletion mutagenesis. The p53-responsive region was determined by measuring the activity of these promoter constructs in the presence of wild-type p53 using luciferase assays. TransFac software was used to identify potential transcription factors in the p53-responsive region. The effect of overexpression of p53 and FOXM1 on MELK expression was examined through Western blotting analysis. Using co-immuno precipitation (Co-IP) studies, we determined the protein-protein interactions between wild-type p53 and FOXM1. The effect of wild-type on FOXM1 recruitment was determined using chromatin immuno precipitation (ChIP) assays. Results: Through promoter deletion analysis, we identified that deletion of the region between -1.69kb to -0.69kb in MELK promoter significantly reduced p53-dependent suppressive effect on MELK promoter activity. Screening this p53-responsive region using TransFac software, we identified a binding site for FOXM1, a previously reported MELK transcription factor. Similar to the regulation of MELK by wild-type p53, overexpression of wild-type p53 repressed FOXM1 and knockdown of wild-type p53 increased FOXM1 expression. Overexpression of FOXM1 increased MELK expression in p53-null and p53-mutant cells but not in p53 wild-type cells. Co-IP studies revealed that wild-type p53 interacts with FOXM1. ChIP studies revealed that overexpression of wild-type p53 significantly reduced the recruitment of FOXM1 to MELK promoter in TNBC cells. Conclusion: These studies elucidated the regulation of MELK expression by wild-type p53 and demonstrated that wild-type p53 represses MELK expression by inhibiting expression and recruitment of FOXM1 to the MELK promoter. In this study, we identified a novel mechanism for upregulation of MELK expression in TNBC, which is associated with poor prognosis of breast cancer patients. 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, Jonathan Shepherd, Dekuang Zhao, Yanxia Ma, Abhijit Mazumdar, Powel H. Brown. Wild-type p53 represses MELK expression by blocking the recruitment of FOXM1 to MELK promoter [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2532.

The human T-cell leukemia virus type-1 p30II protein activates p53 and induces the TIGAR and suppresses oncogene-induced oxidative stress during viral carcinogenesis

In normal cells, aberrant oncogene expression leads to the accumulation of cytotoxic metabolites, including reactive oxygen species (ROS), which can cause oxidative DNA-damage and apoptosis as an intrinsic barrier against neoplastic disease. The c-Myc oncoprotein is overexpressed in many lymphoid cancers due to c-myc gene amplification and/or 8q24 chromosomal translocations. Intriguingly, p53 is a downstream target of c-Myc and hematological malignancies, such as adult T-cell leukemia/lymphoma (ATL), frequently contain wildtype p53 and c-Myc overexpression. We therefore hypothesized that p53-regulated pro-survival signals may thwart the cell's metabolic anticancer defenses to support oncogene-activation in lymphoid cancers. Here we show that the Tp53-induced glycolysis and apoptosis regulator (TIGAR) promotes c-myc oncogene-activation by the human T-cell leukemia virus type-1 (HTLV-1) latency-maintenance factor p30II, associated with c-Myc deregulation in ATL clinical isolates. TIGAR prevents the intracellular accumulation of c-Myc-induced ROS and inhibits oncogene-induced cellular senescence in ATL, acute lymphoblastic leukemia, and multiple myeloma cells with elevated c-Myc expression. Our results allude to a pivotal role for p53-regulated antioxidant signals as mediators of c-Myc oncogenic functions in viral and non-viral lymphoid tumors.

Inhibition of autophagy as a treatment strategy for p53 wild-type acute myeloid leukemia

Here we have explored whether inhibition of autophagy can be used as a treatment strategy for acute myeloid leukemia (AML). Steady-state autophagy was measured in leukemic cell lines and primary human CD34+ AML cells with a large variability in basal autophagy between AMLs observed. The autophagy flux was higher in AMLs classified as poor risk, which are frequently associated with TP53 mutations (TP53mut), compared with favorable- and intermediate-risk AMLs. In addition, the higher flux was associated with a higher expression level of several autophagy genes, but was not affected by alterations in p53 expression by knocking down p53 or overexpression of wild-type p53 or p53R273H. AML CD34+ cells were more sensitive to the autophagy inhibitor hydroxychloroquine (HCQ) than normal bone marrow CD34+ cells. Similar, inhibition of autophagy by knockdown of ATG5 or ATG7 triggered apoptosis, which coincided with increased expression of p53. In contrast to wild-type p53 AML (TP53wt), HCQ treatment did not trigger a BAX and PUMA-dependent apoptotic response in AMLs harboring TP53mut. To further characterize autophagy in the leukemic stem cell-enriched cell fraction AML CD34+ cells were separated into ROSlow and ROShigh subfractions. The immature AML CD34+-enriched ROSlow cells maintained higher basal autophagy and showed reduced survival upon HCQ treatment compared with ROShigh cells. Finally, knockdown of ATG5 inhibits in vivo maintenance of AML CD34+ cells in NSG mice. These results indicate that targeting autophagy might provide new therapeutic options for treatment of AML since it affects the immature AML subfraction.

Abstract AP16: THE CRITICAL ROLE OF P53–TWIST1 AXIS IN OVARIAN CANCER METASTASIS

Abstract BACKGROUND: Tumor metastasis is the primary cause of mortality in patients with advanced ovarian cancer. The molecular mechanism underlying metastasis is still not clear. Twist1 is a transcriptional factor that promotes epithelial-mesenchymal transition (EMT) and enhances the migration and tumor-initiation of ovarian cancer cells. It also has been associated with cancer stem cells and chemoresistance. The purpose of our study is to better understand the regulation of Twist1 and to identify molecular pathways that can be targeted in order to control metastatic diseases. Previous studies have shown an interaction between Twist1 and the tumor suppressor p53. We hypothesize that through their interaction, p53 promotes the proteasomedependent degradation of Twist1 and consequently inhibits EMT in epithelial ovarian cancer cells. p53 mutations, which are common in advanced ovarian cancer patients, may impair p53's ability to inhibit Twist1, leading to enhanced EMT and metastasis. METHOD: In ubiquitin assay p53, Twist1, Pirh2, and Ubiquitin were overexpressed in HEK293T or epithelial ovarian cancer (EOC) cells by transfection. Coimmunoprecipitation and western blotting were used to detect protein interaction and ubiquitination. Protein lysate were prepared from 25 tumor samples (12 ovarian tumor, 10 ovarian tumor metastases, and 2 fallopian tube tumors) for western blotting. Plasmid constructs containing different Twist1 mutants were created to select the degradation resistant mutation. ΔC-Twist1 (C-terminal deletion) was used in in vivo study. RESULT: 1) In EOC cells, proteasome-dependent degradation suppressed Twist1 protein. During in vitro EMT, Twist1 and p53 protein levels negatively correlated. In ovarian cancer cells, wild type p53 overexpression reduced Twist1 protein level and enhances the ubiquitination of Twist1 without affecting Twist1 mRNA level. Mutant p53 (R175H, R148W, and R273H) failed to enhance Twist1 degradation. E3 ligase, Pirh2, formed complex with p53 and Twist1 to induce Twist1 degradation. 2) ΔC-Twist1 was resistant to p53-regulated degradation. In xenograft mouse model, EOC cells overexpressing ΔC-Twist1 could form more aggressive tumors with increased numbers of metastatic lesions than wild type Twist1 group. 3) In 25 ovarian tumor samples, 11 tumors with high levels of Twist1 had either a p53 mutation or very low levels of wild type p53. Nine samples with high levels of wild type p53 all showed low or no Twist1 expression. CONCLUSION: Our data revealed a mechanism by which Twist1 is regulated through p53-promoted proteasome-dependent degradation. p53 facilitates the formation of a Twist1-p53-Pirh2 complex and the Pirh2-mediated Twist1 degradation. Our data also demonstrated that three hotspot p53 mutants failed to promote Twist1 degradation, leading to the stabilization of Twist1 and the induction of EMT. This mechanism may be critical for controlling metastasis of ovarian tumors. The newly discovered role of Pirh2 as an E3 ligase in mediating Twist1 ubiquitination and degradation has expanded the known spectrum of Pirh2 function. In addition to providing new insights into metastatic process at the molecular and cellular levels, our data suggest a signaling pathway that can potentially be used to develop new prognostic markers and therapeutic targets to inhibit Twist1 and control ovarian cancer metastasis. Citation Format: Yang Yang-Hartwich, Roslyn Tedja, Jamie Bingham, Marta Gurrea Soteras, Ayesha B. Alvero, Gil Mor. THE CRITICAL ROLE OF P53–TWIST1 AXIS IN OVARIAN CANCER METASTASIS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP16.

Tumor suppressor protein p53 exerts negative transcriptional regulation on human sodium iodide symporter gene expression in breast cancer.

Aberrant expression of human sodium iodide symporter (NIS) in breast cancer (BC) is well documented but the transcription factors (TF) regulating its aberrant expression is poorly known. We identify the presence of three p53 binding sites on the human NIS promoter sequence by conducting genome-wide TF analysis, and further investigate their regulatory role. The differences in transcription and translation were measured by real-time PCR, luciferase reporter assay, site-directed mutagenesis, in vivo optical imaging, and chromatin immunoprecipitation. The relation of NIS and p53 in clinical samples was judged by TCGA data analysis and immunohistochemistry. Overexpression of wild-type p53 as a transgene or pharmacological activation by doxorubicin drug treatment shows significant suppression of NIS transcription in multiple BC cell types which also results in lowered NIS protein content and cellular iodide intake. NIS repression by activated p53 is further confirmed by non-invasive bioluminescence imaging in live cell and orthotropic tumor model. Abrogation of p53-binding sites by directional mutagenesis confirms reversal of transcriptional activity in wild-type p53-positive BC cells. We also observe direct binding of p53 to these sites on the human NIS promoter. Importantly, TCGA data analysis of NIS and p53 co-expression registers an inverse relationship between the two candidates. Our data for the first time highlight the role of p53 as a negative regulator of functional NIS expression in BC, where the latter is a potential targeted radioiodine therapy candidate. Thus, the study provides an important insight into prospective clinical application of this approach that may significantly impact the patient with mutant versus wild-type p53 profile.

Synergistic targeting of malignant pleural mesothelioma cells by MDM2 inhibitors and TRAIL agonists.

Malignant Pleural Mesothelioma (MPM) is a chemoresistant tumor characterized by low rate of p53 mutation and upregulation of Murine Double Minute 2 (MDM2), suggesting that it may be effectively targeted using MDM2 inhibitors. In the present study, we investigated the anticancer activity of the MDM2 inhibitors Nutlin 3a (in vitro) and RG7112 (in vivo), as single agents or in combination with rhTRAIL.In vitro studies were performed using MPM cell lines derived from epithelioid (ZL55, M14K), biphasic (MSTO211H) and sarcomatoid (ZL34) MPMs. In vivo studies were conducted on a sarcomatoid MPM mouse model.In all the cell lines tested (with the exception of ZL55, which carries a biallelic loss-of-function mutation of p53), Nutlin 3a enhanced p21, MDM2 and DR5 expression, and decreased survivin expression. These changes were associated to cell cycle arrest but not to a significant induction of apoptosis. A synergistic pro-apoptotic effect was obtained through the association of rhTRAIL in all the cell lines harboring functional p53. This synergistic interaction of MDM2 inhibitor and TRAIL agonist was confirmed using a mouse preclinical model. Our results suggest that the combined targeting of MDM2 and TRAIL might provide a novel therapeutic option for treatment of MPM patients, particularly in the case of sarcomatoid MPM with MDM2 overexpression and functional inactivation of wild-type p53.

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.

The PRR11-SKA2 Bidirectional Transcription Unit Is Negatively Regulated by p53 through NF-Y in Lung Cancer Cells.

We previously identified proline-rich protein 11 (PRR11) as a novel cancer-related gene that is implicated in the regulation of cell cycle and tumorigenesis. Our recent study demonstrated that PRR11 and its adjacent gene, kinetochore associated 2 (SKA2), constitute a classic head-to-head gene pair that is coordinately regulated by nuclear factor Y (NF-Y). In the present study, we further show that the PRR11-SKA2 bidirectional transcription unit is an indirect target of the tumor suppressor p53. A luciferase reporter assay revealed that overexpression of wild type p53, but not mutant p53, significantly represses the basal activity and NF-Y mediated transactivation of the PRR11-SKA2 bidirectional promoter. Deletion and mutation analysis of the PRR11-SKA2 promoter revealed that p53-mediated PRR11-SKA2 repression is dependent on the presence of functional NF-Y binding sites. Furthermore, a co-immunoprecipitation assay revealed that p53 associates with NF-Y in lung cancer cells, and a chromatin immunoprecipitation assay showed that p53 represses PRR11-SKA2 transcription by reducing the binding amount of NF-Y in the PRR11-SKA2 promoter region. Consistently, the ability of p53 to downregulate PRR11-SKA2 transcription was significantly attenuated upon siRNA-mediated depletion of nuclear factor Y subunit beta (NF-YB). Notably, lung cancer patients with lower expression of either PRR11 or SKA2 along with wild type p53 exhibited the best overall survival compared with others with p53 mutation and/or higher expression of either PRR11 or SKA2. Taken together, our results demonstrate that p53 negatively regulates the expression of the PRR11-SKA2 bidirectional transcription unit through NF-Y, suggesting that the inability to repress the PRR11-SKA2 bidirectional transcription unit after loss of p53 might contribute to tumorigenesis.

Abstract LB-352: p53-promoted Twist1 degradation inhibits EMT in ovarian cancer cells

Abstract Background: Tumor metastasis is the primary cause of mortality in patients with advanced ovarian cancer. The molecular mechanism behind ovarian cancer metastasis is still not clear. Twist1 is associated with the increased ability of migration and tumor-initiation of ovarian cancer cells; therefore, understanding the regulation of Twist is critical for the prevention of metastatic disease. Previous studies have shown an interaction between Twist1 and the tumor suppressor p53. We propose that p53 functions as a normal inhibitor of Twist1. We hypothesize that through their interaction, p53 promotes the proteasome-dependent degradation of Twist1 and consequently inhibits epithelial-mesenchymal transition (EMT) in epithelial ovarian cancer cells. p53 mutations, which are common in advanced ovarian cancer patients, may impair p53's ability to inhibit Twist1, leading to enhanced EMT and metastasis. Method: p53, Twist1, Pirh2, and Ubiquitin were overexpressed in HEK293T or epithelial ovarian cancer (EOC) cells by transfection. Co-immunoprecipitation and western blotting were used to detect protein interaction and ubiquitination. Protein lysate were prepared from 25 tumor samples (12 ovarian tumor, 10 ovarian tumor metastases, and 2 fallopian tube tumors) for western blotting. Result: Wild type p53 overexpression reduces Twist1 protein level and enhances the ubiquitination of Twist1 without affecting Twist1 mRNA level. Mutant p53 (R175H, R148W, and R273H) failed to regulate Twist1 degradation. E3 ligase, Pirh2, forms complex with p53 and Twist1 to induce Twist1 degradation. In EOC cells, Twist1 is suppressed by proteasome-dependent degradation. During in vitro EMT, Twist1 and p53 protein levels negatively correlated. In 25 ovarian tumor samples, 11 tumors with high levels of Twist1 had either a p53 mutation or very low levels of wild type p53. The 9 samples with high levels of wild type p53 all showed low or no Twist1 expression. Conclusion: Our data revealed a mechanism by which a crucial EMT inducer, Twist1, is regulated by wild type p53 through enhancing its proteasome-dependent degradation. p53 promotes the formation of a Twist1-p53-Pirh2 complex and facilitates Pirh2-mediated Twist1 degradation. Our data also demonstrated that three hotspot p53 mutants failed to promote Twist1 degradation, leading to the stabilization of Twist1 and the induction of EMT. This mechanism may be critical for controlling metastasis of ovarian tumors. The newly discovered role of Pirh2 as an E3 ligase in mediating Twist1 ubiquitination and degradation has expanded the known spectrum of Pirh2 function. In addition to providing new insights into metastatic process at the molecular and cellular levels, our data suggest a signaling pathway that can potentially be used to develop new prognostic markers and therapeutic targets to inhibit Twist1 and control ovarian cancer metastasis. Citation Format: Yang Yang-Hartwich, Roslyn Tedja, Jamie Bingham, Marta Gurrea Soteras, Ayesha B. Alvero, Gil Mor. p53-promoted Twist1 degradation inhibits EMT in ovarian 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 LB-352.

Overexpression of p53 activated by small activating RNA suppresses the growth of human prostate cancer cells.

Previous research has reported that a particular double-stranded RNA, named dsP53-285, has the capacity to induce expression of the tumor suppressor gene TP53 in chimpanzee cells by targeting its promoter. Usually, it is the wild-type p53 protein, rather than mutants, which exhibits potent cancer-inhibiting effects. In addition, nonhuman primates, such as chimpanzees, share almost identical genome sequences with humans. This prompted us to speculate whether dsP53-285 can trigger wild-type p53 protein expression in human prostate cancer (PCa) cells and consequently suppress cell growth. The human PCa cell lines LNCaP and DU145 were transfected with dsP53-285 for 72 hours. Compared with the dsControl and mock transfection groups, expression of both p53 messenger RNA and p53 protein was significantly enhanced after dsP53-285 transfection, and this enhancement was followed by upregulation of p21, which indirectly indicated that dsP53-285 induced wild-type p53 expression. Moreover, overexpression of wild-type p53 mediated by dsP53-285 downregulated the expression of Cyclin D1 and cyclin-dependent kinase 4/6, thereby inducing PCa cell cycle arrest in G0/G1 phase and then inhibiting cell proliferation and clonogenicity. More importantly, dsP53-285 suppressed PCa cells mainly by modulating wild-type p53 expression. In conclusion, our study provides evidence that dsP53-285 can significantly stimulate wild-type p53 expression in the human PCa cell lines LNCaP and DU145 and can exert potent antitumor effects.

P53 overexpression increases chemosensitivity in multidrug-resistant osteosarcoma cell lines.

Multidrug resistance (MDR) is a major obstacle to the successful treatment of osteosarcoma with chemotherapy. Effectiveness of cancer therapy correlates with the ability to induce a p53-dependent apoptotic response. p53 is a tumor suppressor gene that is mutated in 22% of osteosarcomas. While impaired p53 has been implicated in the oncogenesis of osteosarcoma, it is unclear whether overexpression of wild-type p53 can increase chemosensitivity in MDR osteosarcoma cells. We transfected a plasmid encoding the wild-type p53 gene to MDR osteosarcoma cell lines, which have different p53 statuses, U-2OSR2 with wild-type p53 (Wt-p53) and KHOSR2 with mutant p53 (Mt-p53), and determined the effect of p53 overexpression on chemosensitivities. Both of the U-2OSR2 and KHOSR2 cell lines displayed similar trends in p53-induced drug sensitivities. However, it seems that the impact of p53 overexpression is different based on the differential intrinsic p53 status in these cell lines. In the KHOSR2 cell line (Mt-p53), overexpression of p53 up-regulates the expression of pro-apoptotic protein p21 and Bax, while in the U-2OSR2 cell line (Wt-p53), overexpression of p53 down-regulates IGF-1r expression significantly. These results demonstrated that transfection of wild-type p53 increases chemosensitivity either through inhibiting IGF-1r or through increasing the expression of pro-apoptotic proteins p21 and Bax in human MDR osteosarcoma cell lines.

Prognostic and biological significance of survivin expression in patients with diffuse large B-cell lymphoma treated with rituximab-CHOP therapy

Survivin, a member of the inhibitor of apoptosis protein family, is overexpressed in a variety of human neoplasms. The prognostic significance of survivin expression in diffuse large B-cell lymphoma patients treated with rituximab plus cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) is unclear. We used standard immunohistochemistry methods to quantify survivin expression in 463 patients with de novo diffuse large B-cell lymphoma who received the R-CHOP. Of the 463 patients, 269 (58%) had survivin overexpression with a cutoff of >25%, associated with an International Prognostic Index score of >2 (P=0.015), disease in ≥2 extranodal sites (P=0.011), and a high Ki-67 index (P<0.0001). Among patients with activated B cell-like disease, the overall survival rate of survivin-positive patients was significantly lower than that of survivin-negative patients (P=0.033); multivariate analysis confirmed that in these patients, survivin overexpression was an independent prognostic factor for survival. Among patients with wild-type p53 overexpression, the overall survival and progression-free survival rates of the survivin-positive group were significantly lower than those of the survivin-negative group (P=0.035 and P=0.04 respectively). In STAT3-positive patients, survivin overexpression was associated with significantly better survival. Among patients with activated B cell-like disease, survivin-positive compared with survivin-negative groups had significantly different gene expression signatures, including genes involved in mitosis or tumor cell proliferation. Our results indicate that survivin is an independent prognostic factor for poor outcome in patients with activated B cell-like disease treated with the R-CHOP regimen, and patients with survivin-positive activated B cell-like diffuse large B-cell lymphoma seem to benefit less from this treatment and may require additional novel agents.

Elevated p53 promotes the processing of miR-18a to decrease estrogen receptor-α in female hepatocellular carcinoma.

The estrogen pathway has long been implicated as a tumor protector in female hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). Our previous study identified that estrogen receptor alpha (ERα) protein is downregulated in 60% of female HCC cases, via a miR-18a elevation mediated suppression of ERα translation. This study aims to delineate the mechanism underlying the upregulation of miR-18a in female HCC. The analysis of 77 female HCC specimens revealed that miR-18a levels were associated with pre-miR-18a rather than pri-miR-18a levels, suggesting an enhanced processing of pri- to pre-miR-18a. Among a panel of factors involved in microRNA processing, p53 was identified as a novel regulator for miR-18a maturation process. Knockdown of p53 by si-RNA decreased the level of miR-18a, whereas overexpression of either wild-type or mutant p53 increased its level. The association between the elevation of miR-18a and the accumulation of p53, mainly caused by somatic mutations, was confirmed in the clinical specimens of HBV-related female HCC. By analyzing the association with clinicopathological features, activation of this p53/miR-18a pathway mainly occurs in younger or noncirrhosis female HCC patients and associated with a trend of worse overall survival. Therefore, this study demonstrated a novel function of elevated/mutant p53 in regulating the amount of ERα protein through its promoting the biogenesis of miR-18a, which could lead to decrease the tumor-protective function of the estrogen pathway in female hepatocarcinogenesis.