Knockdown Of Mutant P53 Research Articles

Abstract P5-10-03: Mutant p53 and ERK1/2 MAPK cooperate with the production of TNBC inflammatory secretome

Abstract TP53 is the most frequently mutated gene in most types of human cancer, including breast cancer. The triple-negative breast cancer (TNBC) subtype in particular displays TP53 mutation in approximately 80% of patients. Unlike other breast cancer subtypes (e.g., ER/PR-positive or HER2-positive), TNBC patients currently lack an approved highly effective targeted therapy. Notably, most TNBC acquires TP53 mutations, which, in addition to the loss of canonical p53 functions, can result in gain-of-function, activating different cellular mechanisms involved in tumor phenotypes such as proliferation, metastasis, invasiveness, and angiogenesis. Here we evaluate the role of mutant p53 in cancer phenotypes of TNBC cell lines, in particular their inflammatory profile. As expected, loss of p53 protein by small interfering RNA depletion did not show a major impact on the cell viability of MDA-MB231 and Hs578t cells in MTT assay. However, depleting mutant p53 knockdown made MDA-MB231 cells more susceptible to treatment with methyl-methane sulfonate, a genotoxic alkylating agent. Interestingly, cell invasion as measured by the transwell assay demonstrated that depletion of mutant p53 depletion decreased the invasiveness potential of MDA-MB231 and Hs578t cells which was substantiated with decreased migration of MDA-MB231 cells in a scratch assay over 24 h. RNA sequencing of MDA-MB231 and Hs578t cells revealed that mutant p53 knockdown decreased the expression of several constitutively expressed pro-inflammatory cytokines such as IL8, IL6, CXCL2, and CXCL3, but not genes associated with survival to alkylating agents (NRF2 and Endoplasmic reticulum stress markers) or genes typically regulated by wild-type p53 when compared to control-silenced and MMS-treated cells as indicated by Pathway Enrichment Analysis using the Enrich R and DAVID tools. These results were confirmed by ELISA quantification of IL8, IL6, and CXCL2 in MDA-MB231 and Hs578t transfected with two sequences of siRNA targeting mutant p53. On the other hand, RNA sequencing revealed some constitutively expressed genes known to be involved in breast cancer cells malignancy, such as PTGS2 (COX-2 enzyme gene) and MMP1, which were not affected by p53 knockdown. We found that MMP1, and PGE2 (the product of PTGS2/COX-2 enzyme), are upregulated by the ERK1/2 MAPK signaling pathway as determined in MDA-MB231 cells treated with the MEK1/2 inhibitor UO126 and sorafenib. UO126 and sorafenib also decreased IL8 and IL6 production. Furthermore, combined mutant p53 knockdown with MEK/ERK1/2 pathway inhibition caused a more pronounced IL8 and IL6 inhibition when compared to either p53 knockdown or UO126/sorafenib alone, whereas MMP1 and PGE2 levels were only reduced by MEK/ERK1/2 inhibitor treatments. Interestingly, neither mutant p53 knockdown impacted ERK1/2 phosphorylation status nor did UO126/sorafenib alter mutant p53 immunocontent. Reporter assays showed that mutant p53 promotes NFkappaB reporter activation, and MEK/ERK1/2 controls both NFkappaB and AP-1 transcription factors, both associated with the expression of the secretome components evaluated herein. Functional cell assays showed that concomitant inhibition of mutant p53 and MEK/ERK1/2 pathways reduce cell proliferation, invasion, and migration, indicating that mutant p53 protein gain of function cooperates with ERK1/2 MAPK signaling pathway to promote secretome production and malignant phenotypes in TNBC cell models. Citation Format: Raquel Nascimento das Neves, Aparna Gorthi, Alexander James Roy Bishop, Alfeu Zanotto Filho. Mutant p53 and ERK1/2 MAPK cooperate with the production of TNBC inflammatory secretome [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 P5-10-03.

MicroRNA-449a inhibits cell proliferation and migration by regulating mutant p53 in MDA-MB-468 cells.

The present study aimed to investigate the role of microRNA (miR)-449a in the proliferation, migration and apoptosis of MDA-MB-468 breast cancer cells and examine the association between miR-449a and mutant p53 in these cells. Cell proliferation, migration and invasion were examined using a crystal violet staining assay, wound healing scratch assay and Transwell assay, respectively. The expression level of miR-449a and p53 was detected by reverse transcription-quantitative PCR or western blotting. The results indicated that knockdown of mutant p53 suppressed the proliferation and migration of MDA-MB-468 cells by inhibiting the PI3K/AKT/mTOR signaling pathway. In addition, miR-449a suppressed proliferation and migration via downregulation of mutant p53 expression in MDA-MB-468 cells. Therefore, miR-449a may function as a tumor suppressor by regulating p53 expression in breast cancer cells, which may have potential implications in the treatment of patients with triple-negative breast cancer carrying mutant p53.

Mutant p53 induces SIRT3/MnSOD axis to moderate ROS production in melanoma cells

The TP53 tumor suppressor gene is the most frequently altered gene in tumors and mutant p53 isoforms can acquire oncogenic properties referred to as gain-of-function (GOF). In this study, we used wild-type (A375) and mutant p53 (MeWo) melanoma cell lines to assess the regulation of the mitochondrial antioxidant manganese superoxide dismutase (MnSOD) by mutant p53. The effects of mutant p53 were evaluated by qPCR, immunoblotting, enzyme activity assay, cell proliferation assay, reactive oxygen species (ROS) assay after cellular transfection. We demonstrate that mutant p53 induces MnSOD expression, which is recovered by the ROS scavenger N-acetyl-l-cysteine. This suggests MnSOD induction as a defense mechanism of melanoma cells to counterbalance the pro-oxidant conditions induced by mutant p53. We also demonstrate that mutant p53 induces the expression of Sirtuin3 (SIRT3), a major mitochondrial NAD+-dependent deacetylase, stimulating MnSOD deacetylation and enzymatic activity. Indeed, the restoration of SIRT3 reverses MnSOD activity decrease by mutant p53 knock-down. Finally, MnSOD knock-down further enhances mutant p53-mediated ROS increase, counteracting mutp53-dependent cell hyperproliferation. This indicates that SIRT3 and MnSOD act to maintain ROS levels controlled to promote cell proliferation and survival, providing new therapeutic opportunities to be further considered for clinical studies in cancer patients bearing mutant TP53 gene.

Abstract 5296: DNAJA1 promotes cancer progression by stabilizing mutant p53

Abstract The tumor suppressor p53 is mutated in approximately 50% of human cancers. Majority of them are missense mutations, which results in accumulation of dysfunctional p53 proteins in tumors. Increasing evidence suggests that accumulation of mutant p53 is crucial for its oncogenic gain-of-function activities, such as proliferation, metastasis, and chemotherapy resistance. Previous work from our lab has revealed that DNAJA1, a HSP40 family member, specifically binds to and stabilizes conformational/misfolded mutant p53. DNAJA1 downregulation induces ubiquitination and proteasomal degradation of conformational p53 mutants, but does not affect the levels of wild-type p53 or DNA contact mutants. Based on these observations, we hypothesize that DNAJA1 promotes cancer progression in a manner dependent on conformational/misfolded mutant p53. To test this hypothesis, we examined the effects of DNAJA1 deletion/knockdown on malignant properties of head and neck squamous cell carcinoma (HNSCC) cells. Genetic deletion/knockdown of DNAJA1 resulted in reduced proliferation, colony formation, migration, and orthotopic tumor growth of HNSCC cells expressing conformational mutant p53, but not DNA contact mutant p53. These cellular phenotypes induced by DNAJA1 knockdown/knockout were substantially rescued by overexpression of DNAJA1 or mutant p53. We also conducted RNA sequencing analyses using HN31 cells (control, DNAJA1 knockdown or mutant p53 knockdown). Notably, 77.2% of downregulated genes by p53 knockdown were overlapped with genes downregulated by DNAJA1 knockdown. Moreover, genes involved in cancer-related pathways, including adhesion and actin cytoskeleton organization, were commonly downregulated. These results strongly suggest that DNAJA1 promotes HNSCC progression mostly dependent on conformational mutant p53. Our results may also suggest that DNAJA1 could be a novel therapeutic target for cancers carrying conformational/misfolded mutant p53. Citation Format: Tomoo Iwakuma. DNAJA1 promotes cancer progression by stabilizing mutant p53 [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 5296.

Mutant TP53 modulates metastasis of triple negative breast cancer through adenosine A2b receptor signaling.

PurposeThe identification of genes with synthetic lethality in the context of mutant TP53 is a promising strategy for the treatment of basal-like triple negative breast cancer (TNBC). This study investigated regulators of mutant TP53 (R248Q) in basal-like TNBC and their impact on tumorigenesis.Experimental DesignTNBC cells were analyzed by RNA-seq, and synthetic-lethal shRNA knock-down screening, to identify genes related to the expression of mutant TP53. A tissue microarray of 232 breast cancer samples, that included 66 TNBC cases, was used to assess clinicopathological correlates of tumor protein expression. Functional assays were performed in vitro and in vivo to assess the role of ADORA2B in TNBC.ResultsTranscriptome profiling identified ADORA2B as up-regulated in basal-like TNBC cell lines with R248Q-mutated TP53, with shRNA-screening suggesting the potential for a synthetic-lethal interaction between these genes. In clinical samples, ADORA2B was highly expressed in 39.4% (26/66) of TNBC patients. ADORA2B-expression was significantly correlated with ER (P < 0.01), PgR (P = 0.027), EGFR (P < 0.01), and tumor size (P = 0.037), and was an independent prognostic factor for outcome (P = 0.036). In line with this, ADORA2B-transduced TNBC cells showed increased tumorigenesis, and ADORA2B knockdown, along with mutant p53 knockdown, decreased metastasis both in vitro and in vivo. Notably, the cytotoxic cyclic peptide SA-I suppressed ADORA2B expression and tumorigenesis in TNBC cell lines.ConclusionsADORA2B expression increases the oncogenic potential of basal-like TNBC and is an independent factor for poor outcome. These data suggest that ADORA2B could serve as a prognostic biomarker and a potential therapeutic target for basal-like TNBC.

Proteomic Signature of PDAC Cells with Mutant p53

The p53 tumor suppressor plays a crucial role in the prevention of oncogenic transformation through the removal or enduring growth arrest of potentially neoplastic cells. Upon cellular insults, activated p53 serves as a sequence‐specific transcription factor to control the expression of specific genes, thereby stimulating various biological processes. In contrast to the tumor suppressive roles of wild‐type p53, mutant p53 proteins have been shown to support cancer progression by enhancing the ability of cancer cells to invade and metastasize and to confer chemo‐resistance. These remarks demonstrate that mutant p53 possesses gain‐of‐function (GOF) properties that promote oncogenesis. Pancreatic ductal adenocarcinoma (PDAC) is a very aggressive disease with a five‐year survival of less than 5%. The typical features of PDAC, as high genomic instability, chemo‐resistance and invasiveness, constitute also the main peculiarities that cancer cells acquire when the tumor suppressor TP53 gene undergoes GOF mutations, which have been recognized in about 50–75% of PDAC patients. A better understanding of the functional communication between subpopulations of cells within a tumor is essential for the development of effective cancer treatment strategies. Tumor metastasis and invasion are strongly dependent on the composition of the extracellular medium, which is itself affected by the release of proteins by the tumor cells. Mutant p53 proteins are able to influence the secretion of enzymes involved in the modulation of extracellular matrix components, but also to alter the secretion of inflammatory cytokines and to increase the extracellular acidification and to regulate the crosstalk between cancer and stromal cells.The objective of this research was to recognize a specific signature of biomarkers secreted by PDAC cells carrying GOF mutant p53. To state the direct involvement of mutant p53, transfection of siRNAp53 to specifically knock‐down (KD) the endogenous mutant p53 was performed in both wt and mutant p53 PDAC cell lines. To confirm the results the overexpression of mutant p53 was also performed. Using the shotgun proteomics approach and the sequential window acquisition of all theoretical fragment‐ion spectra (SWATH) we were able to quantify hundreds of secreted proteins and to identify potential biomarkers of mutant p53. Furthermore the specific mRNA expression of selected biomarkers was investigated by qRT‐PCR assay.We discovered a number of common proteins having opposite regulation after overexpression and knock‐down of mutant p53. These proteins might constitute a sort of secreted signature driven by the hot‐spot mutant p53 in PDAC and will be validated in serum samples of PDAC patients having WT or mutant TP53 gene. These data might also suggest the identification of targeted therapies specifically addressed to inhibit growth of PDACs carrying oncogenic mutant p53, which are strongly resistant to traditional chemotherapies.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mutant p53 stimulates cell invasion through an interaction with Rad21 in human ovarian cancer cells

Missense mutations of TP53 are extremely common, and mutant p53 accumulation and gain-of-function play crucial roles in human ovarian cancer. Here, we investigated the role of mutant p53 in cell migration and invasion as well as its underlying molecular mechanisms in human ovarian cancer cells. Overexpression of mutant p53 significantly increased migration and invasion in p53-null SKOV3 cells. In contrast, knockdown of mutant p53 significantly compromised mutant p53-induced cell migration and invasion. Microarray analysis revealed that several migration/invasion-related genes, including S1PR1 (Sphingosine-1-phosphate receptor 1) and THBS1 (Thrombospodin 1), were significantly upregulated in SKOV3 cells that overexpressed mutant p53-R248 (SKOV3R248). We found that Rad21 is involved in the transcriptional regulation of the migration/invasion-related genes induced by mutant p53-R248. Knockdown of Rad21 significantly attenuated the mutant p53-R248-induced invasion and the expressions of S1PR1 and THBS1. Moreover, co-immunoprecipitation and chromatin immunoprecipitation assays revealed that mutant p53 interacts with Rad21 and binds to the Rad21-binding elements in the S1PR1 and THBS1 genes. Finally, downregulation of S1PR1 significantly attenuated the invasion driven by mutant p53-R248. These novel findings reveal that mutant p53-R248 maintains gain-of-function activity to stimulate cell invasion and induces the related gene expressions through an interaction with Rad21 in human ovarian cancer cells.

Depletion of runt-related transcription factor 2 (RUNX2) enhances SAHA sensitivity of p53-mutated pancreatic cancer cells through the regulation of mutant p53 and TAp63.

Suberoylanilide hydroxamic acid (SAHA) represents one of the new class of anti-cancer drugs. However, multiple lines of clinical evidence indicate that SAHA might be sometimes ineffective on certain solid tumors including pancreatic cancer. In this study, we have found for the first time that RUNX2/mutant p53/TAp63-regulatory axis has a pivotal role in the determination of SAHA sensitivity of p53-mutated pancreatic cancer MiaPaCa-2 cells. According to our present results, MiaPaCa-2 cells responded poorly to SAHA. Forced depletion of mutant p53 stimulated SAHA-mediated cell death of MiaPaCa-2 cells, which was accomapanied by a further accumulation of γH2AX and cleaved PARP. Under these experimental conditions, pro-oncogenic RUNX2 was strongly down-regulated in mutant p53-depleted MiaPaCa-2 cells. Surprisingly, RUNX2 silencing augmented SAHA-dependent cell death of MiaPaCa-2 cells and caused a significant reduction of mutant p53. Consistent with these observations, overexpression of RUNX2 in MiaPaCa-2 cells restored SAHA-mediated decrease in cell viability and increased the amount of mutant p53. Thus, it is suggestive that there exists a positive auto-regulatory loop between RUNX2 and mutant p53, which might amplify their pro-oncogenic signals. Intriguingly, knockdown of mutant p53 or RUNX2 potentiated SAHA-induced up-regulation of TAp63. Indeed, SAHA-stimulated cell death of MiaPaCa-2 cells was partially attenuated by p63 depletion. Collectively, our present observations strongly suggest that RUNX2/mutant p53/TAp63-regulatory axis is one of the key determinants of SAHA sensitivity of p53-mutated pancreatic cancer cells.

Novel siRNA formulation to effectively knockdown mutant p53 in osteosarcoma.

ObjectivesThe tumor suppressor p53 plays a crucial role in the development of osteosarcoma. The primary objective of this study is to develop and optimize lipid based nanoparticle formulations that can carry siRNA and effectively silence mutant p53 in 318–1, a murine osteosarcoma cell line.MethodsThe nanoparticles were composed of a mixture of two lipids (cholesterol and DOTAP) and either PLGA or PLGA-PEG and prepared by using an EmulsiFlex-B3 high pressure homogenizer. A series of studies that include using different nanoparticles, different amount of siRNAs, cell numbers, incubation time, transfection media volume, and storage temperature was performed to optimize the gene silencing efficiency.Key findingsReplacement of lipids by PLGA or PLGA-PEG decreased the particle size and overall cytotoxicity. Among all lipid-polymer nanoformulations, nanoparticles with 10% PLGA showed highest mutant p53 knockdown efficiency while maintaining higher cell viability when a nanoparticle to siRNA ratio equal to 6.8:0.66 and 75 nM siRNA was used. With long term storage the mutant p53 knockdown efficiency decreased to a greater extent.ConclusionsThis study warrants a future evaluation of this formulation for gene silencing efficiency of mutant p53 in tissue culture and animal models for the treatment of osteosarcoma.

Double gene siRNA knockdown of mutant p53 and TNF induces apoptosis in triple-negative breast cancer cells.

Apoptosis is the major downregulated pathway in cancer. Simultaneous inhibition using specific small interfering RNA (siRNA) of two key player genes, p53 and TNF, is an interesting and feasible strategy when it comes to investigating various molecular pathways and biological processes in triple-negative breast cancer (TNBC), which is one of the most aggressive and therapeutically unresponsive forms of breast cancers. Our present research focuses on evaluating the impact of double p53-siRNA and TNF-siRNA knockdown at a cellular level, and also evaluating cell proliferation, apoptosis, induction of autophagy, and gene expression by using reverse transcription polymerase chain reaction array approaches. Simultaneous inhibition of p53 and TNF in Hs578T TNBC human cell line revealed a panel of up- and downregulated genes involved in apoptosis. Furthermore, the effects of double gene knockdown were validated in a second TNBC cell line, MDA-MB-231, by using reverse transcription polymerase chain reaction TaqMan assay. All our findings help in understanding the functional mechanisms of extrinsic apoptosis, cell signaling pathways, and the mechanisms involved in tumor cell survival, growth, and death in TNBC.

Depletion of pro-oncogenic RUNX2 enhances gemcitabine (GEM) sensitivity of p53-mutated pancreatic cancer Panc-1 cells through the induction of pro-apoptotic TAp63.

Recently, we have described that siRNA-mediated silencing of runt-related transcription factor 2 (RUNX2) improves anti-cancer drug gemcitabine (GEM) sensitivity of p53-deficient human pancreatic cancer AsPC-1 cells through the augmentation of p53 family TAp63-dependent cell death pathway. In this manuscript, we have extended our study to p53-mutated human pancreatic cancer Panc-1 cells. According to our present results, knockdown of mutant p53 alone had a marginal effect on GEM-mediated cell death of Panc-1 cells. We then sought to deplete RUNX2 using siRNA in Panc-1 cells and examined its effect on GEM sensitivity. Under our experimental conditions, RUNX2 knockdown caused a significant enhancement of GEM sensitivity of Panc-1 cells. Notably, GEM-mediated induction of TAp63 but not of TAp73 was further stimulated in RUNX2-depleted Panc-1 cells, indicating that, like AsPC-1 cells, TAp63 might play a pivotal role in the regulation of GEM sensitivity of Panc-1 cells. Consistent with this notion, forced expression of TAp63α in Panc-1 cells promoted cell cycle arrest and/or cell death, and massively increased luciferase activities driven by TAp63-target gene promoters such as p21WAF1 and NOXA. In addition, immunoprecipitation experiments indicated that RUNX2 forms a complex with TAp63 in Panc-1 cells. Taken together, our current observations strongly suggest that depletion of RUNX2 enhances the cytotoxic effect of GEM on p53-mutated Panc-1 cells through the stimulation of TAp63-dependent cell death pathway even in the presence of a large amount of pro-oncogenic mutant p53, and might provide an attractive strategy to treat pancreatic cancer patients with p53 mutations.

Allele-specific silencing of mutant p53 attenuates dominant-negative and gain-of-function activities.

Many p53 hotspot mutants not only lose the transcriptional activity, but also show dominant-negative (DN) and oncogenic gain-of-function (GOF) activities. Increasing evidence indicates that knockdown of mutant p53 (mutp53) in cancer cells reduces their aggressive properties, suggesting that survival and proliferation of cancer cells are, at least partially, dependent on the presence of mutp53. However, these p53 siRNAs can downregulate both wild-type p53 (wtp53) and mutp53, which limits their therapeutic applications. In order to specifically deplete mutp53, we have developed allele-specific siRNAs against p53 hotspot mutants and validated their biological effects in the absence or presence of wtp53. First, the mutp53-specific siRNAs selectively reduced protein levels of matched p53 mutants with minimal reduction in wtp53 levels. Second, downregulation of mutp53 in cancer cells expressing a mutp53 alone (p53mut) resulted in significantly decreased cell proliferation and migration. Third, transfection of mutp53-specific siRNAs in cancer cells expressing both wtp53 and mutp53 also reduced cell proliferation and migration with increased transcripts of p53 downstream target genes, which became further profound when cells were treated with an MDM2 inhibitor Nutlin-3a or a chemotherapeutic agent doxorubicin. These results indicate that depletion of mutp53 by its specific siRNA restored endogenous wtp53 activity in cells expressing both wtp53 and mutp53. This is the first study demonstrating biological effects and therapeutic potential of allele-specific silencing of mutp53 by mutp53-specific siRNAs in cancer cells expressing both wtp53 and mutp53, thus providing a novel strategy towards targeted cancer therapies.

Mutant p53 promotes ovarian cancer cell adhesion to mesothelial cells via integrin β4 and Akt signals.

Missense mutations in the TP53 gene resulting in the accumulation of mutant proteins are extremely common in advanced ovarian cancer, which is characterised by peritoneal metastasis. Attachment of cancer cells to the peritoneal mesothelium is regarded as an initial, key step for the metastatic spread of ovarian cancer. In the present study, we investigated the possible role of a p53 mutant in the mesothelial adhesion of ovarian cancer cells. We found that OVCAR-3 cells with the R248 TP53 mutation (p53R248) were more adhesive to mesothelial Met5A cells than were A2780 cells expressing wild-type p53. In addition, ectopic expression of p53R248 in p53-null SKOV-3 cells significantly increased adhesion to Met5A cells. Knockdown of mutant p53 significantly compromised p53R248-induced cell adhesion to Met5A cells. Microarray analysis revealed that several adhesion-related genes, including integrin β4, were markedly up-regulated, and certain signalling pathways, including PI3K/Akt, were activated in p53R248 transfectants of SKOV-3 cells. Inhibition of integrin β4 and Akt signalling using blocking antibody and the inhibitor LY294002, respectively, significantly attenuated p53R248-mediated ovarian cancer-mesothelial adhesion. These data suggest that the p53R248 mutant endows ovarian cancer cells with increased adhesiveness and that integrin β4 and Akt signalling are associated with the mutation-enhanced ovarian cancer-mesothelial cell adhesion.

The p53R172H mutant does not enhance hepatocellular carcinoma development and progression.

Hepatocellular carcinoma is a highly deadly malignancy, accounting for approximately 800,000 deaths worldwide every year. Mutation of the p53 tumor suppressor gene is a common genetic change in HCC, present in 30% of cases. p53R175H (corresponding to p53R172H in mice) is a hotspot for mutation that demonstrates “prometastatic” gain-of-function in other cancer models. Since the frequency of p53 mutation increases with tumor grade in HCC, we hypothesized that p53R172H is a gain-of-function mutation in HCC that contributes to a decrease in tumor-free survival and an increase in metastasis. In an HCC mouse model, we found that p53R172H/flox mice do not have decreased survival, increased tumor incidence, or increased metastasis, relative to p53flox/flox littermates. Analysis of cell lines derived from both genotypes indicated that there are no differences in anchorage-independent growth and cell migration. However, shRNA-mediated knockdown of mutant p53 in p53R172H-expressing HCC cell lines resulted in decreased cell migration and anchorage-independent growth. Thus, although p53 mutant-expressing cells and tumors do not have enhanced properties relative to their p53 null counterparts, p53R172H-expressing HCC cells depend on this mutant for their transformation. p53 mutants have been previously shown to bind and inhibit the p53 family proteins p63 and p73. Interestingly, we find that the levels of p63 and p73 target genes are similar in p53 mutant and p53 null HCC cells. These data suggest that pathways regulated by these p53 family members are similarly impacted by p53R172H in mutant expressing cells, and by alternate mechanisms in p53 null cells, resulting in equivalent phenotypes. Consistent with this, we find that p53 null HCC cell lines display lower levels of the TA isoforms of p63 and p73 and higher levels of ΔNp63. Taken together these data point to the importance of p63 and p73 in constraining HCC progression.

Abstract 1551: p53 mutant regulates a network of cellular anoikis through activation of AKT and suppression of BMF

Abstract p53 is the most frequently mutated tumor suppressor gene in human cancers. Unlike other tumor-suppressor genes, p53 mutations mainly occur as missense mutations within the DNA-binding domain, leading to the expression of full-length mutant p53 protein. Mutant p53 proteins not only lose their tumor suppressor function, but may also gain new oncogenic function and promote tumorigenesis through transactivation of new target genes. By using an RNA interference (RNAi) approach, we show that silencing of endogenous p53 R273H contact mutant in MDA-MB-468 cells induced massive apoptosis while no apoptosis was observed in the silencing of the p53 R175H structural mutant in SKBR3 cells. Further analysis shows that the apoptotic effects of mutant p53 knock-down involved activation of caspase-9 and mitochondria depolarisation, suggesting a role for mutant p53 in suppressing the intrinsic apoptotic pathway. Indeed, a BCL-2 family member, BCL-2 modifying factor (BMF), was reproducibly up-regulated following knock-down of mutant p53 in MDA-MB-468 cells while no significant changes was observed in other BCL2 family members. Furthermore, we show that p53 R273H contact mutant, but not p53 R175H conformation mutant, suppressed anoikis by down-regulation of BMF expression. Anoikis is a form of apoptosis induced by cell detachment and is a major mechanism to prevent metastasis. Further analyses using microarray gene profiling and connectivity map analysis show that silencing of mutant p53 in MDA-MB-468 cells generates gene expression signatures characteristic of PI3K inhibitors, suggesting that apoptotic effects of mutant p53 knock-down involved dephosphorylation of AKT, a direct substrate of PI3K. Ectopic expression of the activated form of AKT in MDA-MB-468 completely abrogated the apoptotic effects of mutant p53 knock-down. In summary, our results demonstrate that R273H mutant p53 promotes anoikis resistance in breast cancer cells that express it through suppression of BMF expression by the constitutively activated AKT pathway. Citation Format: Boon Shing Tan, Kai Hung Tiong, Ivan Kok Seng Yap, Rozita Rosli, Soon-Keng Cheong, Chee-Onn Leong. p53 mutant regulates a network of cellular anoikis through activation of AKT and suppression of BMF. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1551. doi:10.1158/1538-7445.AM2014-1551

Abstract LB-84: Mutant p53 enhances the metastatic potential of ovarian cancer cells

Abstract Missense-mutations of TP53 resulting in accumulation of mutant proteins are very frequent in human cancer including ovarian cancer. Aggressive ovarian cancer is characterized by a peritoneal metastasis which involves the adhesion, migration, and invasion of tumor cells to the peritoneal cavity. In the present study, we investigated the effect of p53 GOF mutation (p53R248) on the metastatic potential of human ovarian cancer cells. We found that forced overexpression of p53R248 significantly increased adhesion to mesothelial cell Met5A, migration, and invasion in p53-null SKOV-3 cells. Conversely, knockdown of mutant p53 using siRNA significantly compromised p53R248-induced cell adhesion, migration, and invasion. Microarray analysis revealed that several metastasis-related genes including integrins and S1PR1(Sphingosine-1-phosphate receptor 1), and certain signaling pathway including PI3K/AKT were markedly up-regulated in p53R248 transfectant SKOV-3R248 cells. Inhibition of PI3K/Akt signaling using specific inhibitor significantly attenuated p53R248-mediated mesothelial cell adhesion, migration, and invasion. These finding show for the first time that mutant p53R248 may exert gain-of-function activity to regulate metastasis-related gene expression and the PI3K/Akt singling pathway in ovarian cancer. Citation Format: Ji-Hye Ahn, Jong-Gyu Lee, Jung-Hye Choi. Mutant p53 enhances the metastatic potential of ovarian cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-84. doi:10.1158/1538-7445.AM2014-LB-84

Abstract P3-03-05: Modulation of the metastatic propensity of triple-negative breast cancer cells harboring p53 mutations

Abstract The mutation of p53 is implicated in the increased metastatic potential of triple-negative (ER—/PR—/Her2—) breast cancer (TNBC). The low survival rates of advanced metastatic cancer make it critical not only to understand the mechanisms underlying the functional implications of p53 mutations, but also to identify ways to regulate their impact in cancer cell physiology. A common gain-of-function activity of the mutant p53 protein is the enhancement of cell migration and invasion. We have found that a hydrocarbon-stapled p53 transactivation domain peptide (SAH-p53) designed to disrupt the interaction with p53 negative regulators HDM2 and HDMX also has the ability to inhibit the interaction between mutant p53 (mtp53) and HDM2/HDMX. In addition, SAH-p53 was found to inhibit the migration and invasion of MDA-MB-231 cells (mtp53), while exhibiting no alteration in their proliferative ability. The capacity of SAH-p53 to inhibit MDA-MB-231 cell migration was not altered in the absence of HDMX, suggesting an alternative mechanism of action for SAH-p53. The propensity of MDA-MB-231 cells to migrate and invade through Matrigel in response to transforming growth factor b (TGF-β), epidermal growth factor (EGF), hepatocyte growth factor (HGF), and platelet-derived growth factor (PDGF) was decreased by treatment with SAH-p53. Furthermore, increased concentrations of these growth factors displayed a synergistic effect with SAH-p53 in the inhibition of cell migration, suggesting cross-talk between receptor signaling and SAH-p53 activity. This effect was accompanied by an alteration in stress fiber formation and dissolution of focal adhesions. While the knockdown of mutant p53 in MDA-MB-231 cells significantly decreased migration on its own, inhibition of migration by SAH-p53 was not seen in the EGFR/p53-null MDA-MB-157 cells, suggesting a need for either the p53 or EGFR protein for the inhibitory activity of SAH-p53. Moreover, a SAH-p53-induced decrease in the total levels of EGFR and integrin beta1, together with alterations in actin stability, suggests a defect in receptor recycling, a process required for cell migration known to be modulated by an interaction between mutant p53 and p63. These data show that the use of the SAH-p53 peptide provides a novel avenue to study the molecular interactions that drive metastatic behavior originated by p53 gain-of-function mutations. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-03-05.

Gain-of-function mutant p53-R280K mediates survival of breast cancer cells

Missense mutations in TP53 resulting in the expression of p53-R175H, p53-R273H, or p53-R280K are frequently detected in human breast cancer. Currently, the role of mutant p53-R280K in breast cancer is relatively unknown, and therefore, the present study analyzed the function of mutant p53-R280K in breast cancer cell growth. To this end, we used small interfering RNA to study the role of mutant p53-R280K in MDA-MB-231 cells, which endogenously express the mutant protein. We found that curcumin induced apoptosis in MDA-MB-231 cells and downregulated mutant p53-R280K. We also observed that knockdown of mutant p53 by small interfering RNA induced apoptosis in MDA-MB-231 cells. Curcumin-induced apoptosis was further enhanced by the overexpression of wild-type p53, but was decreased by mutant p53-R280K overexpression. Our findings indicate that mutant p53-R280K has an important role in mediating the survival of triple-negative breast cancer MDA-MB-231 cells. Furthermore, this study suggests mutant p53-R280K could be used as a therapeutic target for breast cancer cells harboring this TP53 missense mutation.

Heat-shock factor 1 both positively and negatively affects cellular clonogenic growth depending on p53 status

HSF1 (heat-shock factor 1) is the master regulator of the heat-shock response; however, it is also activated by cancer-associated stresses and supports cellular transformation and cancer progression. We examined the role of HSF1 in relation to cancer cell clonogenicity, an important attribute of cancer cells. Ectopic expression or HSF1 knockdown demonstrated that HSF1 positively regulated cancer cell clonogenic growth. Furthermore, knockdown of mutant p53 indicated that HSF1 actions were mediated via a mutant p53-dependent mechanism. To examine this relationship more specifically, we ectopically co-expressed mutant p53(R273H) and HSF1 in the human mammary epithelial cell line MCF10A. Surprisingly, within this cellular context, HSF1 inhibited clonogenicity. However, upon specific knockdown of endogenous wild-type p53, leaving mutant p53(R273H) expression intact, HSF1 was observed to greatly enhance clonogenic growth of the cells, indicating that HSF1 suppressed clonogenicity via wild-type p53. To confirm this we ectopically expressed HSF1 in non-transformed and H-Ras(V12)-transformed MCF10A cells. As expected, HSF1 significantly reduced clonogenicity, altering wild-type p53 target gene expression levels consistent with a role of HSF1 increasing wild-type p53 activity. In support of this finding, knockdown of wild-type p53 negated the inhibitory effects of HSF1 expression. We thus show that HSF1 can affect clonogenic growth in a p53 context-dependent manner, and can act via both mutant and wild-type p53 to bring about divergent effects upon clonogenicity. These findings have important implications for our understanding of HSF1's divergent roles in cancer cell growth and survival as well as its disparate effect on mutant and wild-type p53.

Abstract LB-53: Prodigiosin rescues mutant p53 signaling and antitumor effects via p73 upregulation and disruption of mutant p53:p73 interaction.

Abstract Rescue of mutant p53 function is an attractive strategy for cancer therapy development. Using high throughput drug screening, we identified prodigiosin, which can reactivate p53-dependent transcriptional activity in p53 mutant cancer cells. We find that prodigiosin has the therapeutic potential of p53 signaling restoration in a panel of cancer cells with p53 hotspot mutants. Our study has indicated that prodigiosin can up-regulate the expression of p73. However, the function of the increased p73 could be inhibited by interacting with the overexpressed mutant p53 in cancer cells. The immunoprecipitation assay indicates that prodigiosin treatment disrupts the interaction of mutant p53 and p73 in mutant p53-containing SW480 cells. Prodigiosin has been reported to be able to bind to mTOR and inhibit mTOR activity. In order to determine whether prodigiosin disrupts the interaction of mutant p53/p73 complex through mTOR inhibition, we treated SW480 cells with the mTOR inhibitor rapamycin to assess whether mTOR inhibition can disrupt the interaction. The results indicate that prodigiosin disrupts the physical interaction of mutant p53 and p73, but rapamycin does not disrupt this interaction. Western blot confirms that both prodigiosin and rapamycin inhibit the mTOR pathway as indicated by the decrease of phospho-mTOR, phospho-p70 S6 Kinase and phospho-4E-BP1 when SW480 cells are treated with either prodigiosin or rapamycin. The result suggests that prodigiosin disrupts the interaction of mutant p53/p73 complex independent of mTOR inhibition. Furthermore, we examine the role of mutant p53 in the mechanism of p53 restoration by prodigiosin. By the knock-down of mutant p53 by siRNA treatment, we find that prodigiosin-induced p53 activity is increased in mutant p53 knock-down SW480 cells and mutant p53 knock-down SW480 cells are more sensitive to prodigiosin-induced cell death. The result suggests that mutant p53 has the dominant-negative function. Our study implies that the only p73 up-regulation may not have a potent anti-tumor effect in p53 mutant cancers, as overexpressed mutant p53 would be expected to inhibit p73 function by protein-protein interaction. Our study indicates that prodigiosin not only up-regulates p73, but also prevents p73 from interacting with mutant p53. Therefore, the dual effects (up-regulation of p73 and disruption of the interaction between mutant p53 and p73) of prodigiosin make it a very promising candidate in anti-cancer drug development. This work was supported by the NCI Developmental Therapeutics Program through NCI Contract N01-CN-43302. Citation Format: Bo Hong, Antonius Van Den Heuvel, Levy Kopelovich, Wafik S. El-Deiry. Prodigiosin rescues mutant p53 signaling and antitumor effects via p73 upregulation and disruption of mutant p53:p73 interaction. [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 LB-53. doi:10.1158/1538-7445.AM2013-LB-53