Gain Of Oncogenic Function Research Articles

Salvation of the fallen angel: Reactivating mutant p53.

The transcription factor p53 is known as the guardian of the genome for its powerful anti-tumour capacity. However, mutations of p53 that undermine their protein structure, resulting in loss of tumour suppressor function and gain of oncogenic function, have been implicated in more than half of human cancers. The crucial role of mutant forms of p53 in cancer makes it an attractive therapeutic target. A large number of candidates, including low MW compounds, peptides, and nucleic acids, have been identified or designed to rescue p53 mutants and reactivate their anti-tumour capacity through a variety of mechanisms. In this review, we summarize the progress made in the reactivation of mutant forms of p53, focusing on the pharmacological mechanisms of the reactivators of p53 mutants.

Regulators of Oncogenic Mutant TP53 Gain of Function.

The tumor suppressor p53 (TP53) is the most frequently mutated human gene. Mutations in TP53 not only disrupt its tumor suppressor function, but also endow oncogenic gain-of-function (GOF) activities in a manner independent of wild-type TP53 (wtp53). Mutant TP53 (mutp53) GOF is mainly mediated by its binding with other tumor suppressive or oncogenic proteins. Increasing evidence indicates that stabilization of mutp53 is crucial for its GOF activity. However, little is known about factors that alter mutp53 stability and its oncogenic GOF activities. In this review article, we primarily summarize key regulators of mutp53 stability/activities, including genotoxic stress, post-translational modifications, ubiquitin ligases, and molecular chaperones, as well as a single nucleotide polymorphism (SNP) and dimer-forming mutations in mutp53.

Methods to Screen Compounds Against Mutant p53 Misfolding and Aggregation for Cancer Therapeutics.

p53 is a critical tumor suppressor that functions as a transcription factor. Mutations in the TP53 gene are observed in more than 50% of cancer cases worldwide. Several of these mutations lead to a less stable, aggregation-prone protein that accumulates in cancer cells. These mutations are associated with a gain of oncogenic function, which leads to cancer progression. p53 amyloid aggregation is a common feature in most of these mutants; thus, it can be used as a druggable target to reactivate or induce the degradation of p53 and promote a retraction in the aggressive pattern of mutant p53-containing cells. We show here a series of experiments for the screening and validation of new p53 antiamyloid compounds.

Abstract A25: Urokinase plasminogen activator expression is regulated by p53 harboring the lung cancer-specific mutation V157F

Abstract Rationale: Cancers of the lung and bronchus are responsible for nearly 30% of all cancer-related deaths in the US, and mutations in the tumor-suppressor gene TP53 occur in 54% of lung adenocarcinomas and 86% of squamous cell carcinomas. Compared with other cancers, TP53 mutational hotspots in lung cancer more often include missense mutations at codons V157, R158, and A159, with frequencies approaching that of the canonical mutational hotspot R273. The resulting mutant p53 (mutp53) protein often exhibits not only loss of tumor suppressor capability but also gain of oncogenic function (GOF). Our previous work identified a novel, p53-dependent transcriptome including alterations in expression of urokinase plasminogen activator (uPA), a key regulator of the plasminogen activation system. When activated, uPA converts plasminogen to plasmin, which cleaves extracellular matrix components and promotes cell migration. We evaluated uPA expression and cell migration in human lung cancer cell lines expressing wild-type or mutant p53. Methods: Human lung cancer cell lines with wild-type p53 (A549) or with somatic mutations at the endogenous p53 locus (NCI-H1781 and NCI-H2087, V157F; NCI-H441, R158L; NCI-H2110, R158P; NCI-H2009, R273L) were obtained from ATCC. Using RNAi (shRNA and siRNA) cells were depleted of p53 as confirmed by puromycin selection of cells infected with lentivirus, in the case of shRNA. Whole-cell lysates were harvested for protein and RNA purification, and conditioned medium was harvested for measurement of secreted proteins. Immunoblotting and qRT-PCR were used to assess expression of protein and mRNA, respectively. A wound healing assay was performed following siRNA transfection to determine cell migration in cells expressing vs. depleted of wild-type or mutant p53. Results: Silencing of p53 by shRNA and siRNA targeted to TP53 was confirmed by Western blot and qRT-PCR. With p53 depletion, protein and mRNA expression of the uPA pro-enzyme pro-uPA and its activated B- and A-chains increased in the H2087 (V157F mutp53) cell line. No p53-dependent change was seen in uPA expression in H2009 cells (R273L mutp53) despite a similar relative quantity of uPA protein. Secreted uPA also increased as measured in conditioned media from H2087 cells (V157F mutp53). A wound healing assay showed increased migration by mutp53-depleted H2087 cells compared with mutp53-expressing H2087 cells, while A549 cells (wt p53) showed no p53-dependent difference in migration rate. Conclusions: To our knowledge, GOF phenotypes in lung cancer cells carrying V157F mutp53 have not been characterized. We have shown here that mutp53 represses uPA expression in human lung cancer cells with endogenous V157F mutp53, and depletion of p53 in these cells leads to an increase in migration. These findings prompt a reexamination of the signaling pathways associated with uPA. Further study is needed to elucidate the mechanism of the p53-dependent alterations in uPA expression and to establish additional biologic effects of V157F mutp53. Citation Format: Julie A. Barta, Steven B. McMahon. Urokinase plasminogen activator expression is regulated by p53 harboring the lung cancer-specific mutation V157F [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr A25.

Abstract 2500: Identification and characterization of a bipartite nuclear localization signal reveals non-canonical 'oncogenic' role for cytoplasm-restricted ARID1B

Abstract The AT-rich interaction domain-containing protein 1B (ARID1B/ BAF250b) is a component of SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complex that functions as the primary chromatin remodeler during ontogeny and adult life. ARID1B is classified as a tumor suppressor in melanoma as well as in cancers of the uterus, ovary, lung, breast, colon and pancreas. Though human ARID1B is a nuclear protein, the molecular basis of its nuclear import is not understood. Using protein truncation analysis, in silico prediction and sequence comparison within forty three metazoan species, we identified a putative bipartite Nuclear localization signal (NLS) sequence, further confirmed by immunofluorescence analyses and cell fractionation followed by immunoblotting. An immunoprecipitation-mass spectrometry based screen for identifying ARID1B interacting proteins revealed members of the importin family (IPO5, KPNA2 and KPNB1). In addition, ARID1B nuclear import was inhibited by Importazole (general importin β inhibitor). Transcription activation function of the cytoplasm-restricted ARID1B-NLS mutant, as measured by quantifying CDKN1A and CDKN1B transcript and protein levels as well as promoter activity following ectopic expression in cancer cell lines, was significantly compromised when compared to wild type ARID1B. The NLS-mutant was also restricted in its ability to induce senescence. Surprisingly however, cytoplasmic localization of ARID1B appeared to result in an ‘oncogenic' gain of function as it boosted cell proliferation, promoted ERK signaling and activated β-catenin/TCF-LEF function. A detailed analysis of the oncogenic role of cytoplasm-restricted ARID1B is ongoing. A careful scrutiny of various cancer specific somatic mutation databases revealed several mutations located within the ARID1B NLS sequence. In addition, ARID1B immunohistochemistry on breast cancer tissue microarrays (TMAs) revealed significant cytoplasmic localization. These observations suggest possible clinical relevance of cytoplasm-restricted ARID1B. Functional validation of NLS specific mutations and TMA analysis of other cancers is currently underway. Non-canonical oncogenic gain of function due to aberrant cytoplasmic localization has been shown previously for classical tumor suppressors such as pRB, p27, p21 and p53. Our results have revealed a novel ‘oncogenic' facet of ARID1B resulting from its aberrant cytoplasmic localization. Citation Format: Srinivas Animireddy, Padmavathi Kavadipula, Viswakalyan Kotapalli, Swarnalata Gowrishankar, Satish Rao, Murali D. Bashyam. Identification and characterization of a bipartite nuclear localization signal reveals non-canonical 'oncogenic' role for cytoplasm-restricted ARID1B [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 2500.

Catalytic domain mutation in CYLD inactivates its enzyme function by structural perturbation and induces cell migration and proliferation

Tumor suppressor cylindromatosis protein (CYLD), which specifically cleaves lysine 63-linked ubiquitin chain from its substrate molecules, contributes to myriad of important cellular events including cellular differentiation, oncogenesis, DNA repair and cell cycle control. It is a ubiquitously expressed protein, which negatively regulates NF-kB and JNK signaling pathways and mediates caspase dependent apoptosis through RIP1 deubiqutination. Germline mutations in CYLD are associated with a rare, hypertrophic skin cancer, termed Familial Cylindromatosis. Catalogue of Somatic Mutations in Cancer database ensembles accumulating CYLD point mutations in multiple benign and malignant tumors. However, the functional role of CYLD mutations and their association with cancer progression remains elusive. In the present report, we have shown that cancer associated mutations impose structural alteration in CYLD which impairs its binding to K63 ubiquitin chain. Here, we conclude that loss of CYLD catalytic activity potentiates its oncogenic gain of function through increased cell survival and migration.

Plakoglobin restores tumor suppressor activity of p53R175H mutant by sequestering the oncogenic potential of β-catenin.

Tumor suppressor/transcription factor p53 is mutated in over 50% of all cancers. Some mutant p53 proteins have not only lost tumor suppressor activities but they also gain oncogenic functions (GOF). One of the most frequently expressed GOF p53 mutants is Arg175His (p53R175H) with well‐documented roles in cancer development and progression. Plakoglobin is a cell adhesion and signaling protein and a paralog of β‐catenin. Unlike β‐catenin that has oncogenic function through its role in the Wnt pathway, plakoglobin generally acts as a tumor/metastasis suppressor. We have shown that plakoglobin interacted with wild type and a number of p53 mutants in various carcinoma cell lines. Plakoglobin and mutant p53 interacted with the promoter and regulated the expression of several p53 target genes. Furthermore, plakoglobin interactions with p53 mutants restored their tumor suppressor/metastasis activities in vitro. GOF p53 mutants induce accumulation and oncogenic activation of β‐catenin. Previously, we showed that one mechanism by which plakoglobin may suppress tumorigenesis is by sequestering β‐catenin's oncogenic activity. Here, we examined the effects of p53R175H expression on β‐catenin accumulation and transcriptional activation and their modifications by plakoglobin coexpression. We showed that p53R175H expression in plakoglobin null cells increased total and nuclear levels of β‐catenin and its transcriptional activity. Coexpression of plakoglobin in these cells promoted β‐catenin's proteasomal degradation, and decreased its nuclear levels and transactivation. Wnt/β‐catenin targets, c‐MYC and S100A4 were upregulated in p53R175H cells and were downregulated when plakoglobin was coexpressed. Plakoglobin‐p53R175H cells also showed significant reduction in their migration and invasion in vitro.

Mutant p53 cancers reprogram macrophages to tumor supporting macrophages via exosomal miR-1246

TP53 mutants (mutp53) are involved in the pathogenesis of most human cancers. Specific mutp53 proteins gain oncogenic functions (GOFs) distinct from the tumor suppressor activity of the wild-type protein. Tumor-associated macrophages (TAMs), a hallmark of solid tumors, are typically correlated with poor prognosis. Here, we report a non-cell-autonomous mechanism, whereby human mutp53 cancer cells reprogram macrophages to a tumor supportive and anti-inflammatory state. The colon cancer cells harboring GOF mutp53 selectively shed miR-1246-enriched exosomes. Uptake of these exosomes by neighboring macrophages triggers their miR-1246-dependent reprogramming into a cancer-promoting state. Mutp53-reprogammed TAMs favor anti-inflammatory immunosuppression with increased activity of TGF-β. These findings, associated with poor survival in colon cancer patients, strongly support a microenvironmental GOF role for mutp53 in actively engaging the immune system to promote cancer progression and metastasis.

Abstract 73: Gain-of-function mutant p53 promotes the oncogenic potential of head and neck squamous cell carcinoma cell by targeting forkhead transcription factors FOXO3a and FOXM1

Abstract Many mutant p53 proteins exert oncogenic gain-of-function (GOF) properties in promoting cancer cell invasive growth and metastasis, but the mechanisms mediating these functions largely remain elusive. We show here that overexpression of the GOF mutant p53 G245D and other GOF p53 mutants enhances invasive cell growth of p53-deficient head and neck squamous cell carcinoma (HNSCC) UM-SCC-1 cells both in vitro 3D culture and in vivo orthotopic tumor mouse model. We demonstrate that the expression of the oncogenic forkhead transcription factor FOXM1 is upregulated by GOF mutant p53s in UM-SCC-1 cells and tumors. Moreover, we show that overexpression of GOF mutant p53 G245D decreases the inhibitory phosphorylation of FOXO3a, a tumor suppressive forkhead transcription factor, leading to its cytoplasmic accumulation. Consistent with the observation that FOXO3a negatively regulates FOXM1 expression, we demonstrate that overexpression FOXO3a decreases GOF mutant p53-mediated FOXM1 expression, whereas downregulation of FOXO3a partially restores decreased FOXM1 expression due to loss of GOF mutant p53. In support of the roles of FOXO3a-FOXM1 signaling axis in mutant p53's GOF signaling, we further show that overexpression of FOXO3a or downregulation of FOXM1 impairs GOF mutant p53-mediated cell invasion, whereas downregulation of FOXO3a, in part, rescues impaired cell invasion due to downregulated GOF mutant p53. Finally, given that AMP-activated protein kinase (AMPK) directly phosphorylates and promotes FOXO3a function, and that our prior demonstration that GOF mutant p53s inhibit AMPK, our current study establishes that GOF mutant p53-AMPK-FOX3a-FOXM1 signaling is one of important mechanisms through which mutant p53s gain oncogenic functions in HNSCCs. Citation Format: Noriaki Tanaka, Mei Zhao, Jiexin Zhang, Jing Wang, Ge Zhou, Jeffrey N. Myers. Gain-of-function mutant p53 promotes the oncogenic potential of head and neck squamous cell carcinoma cell by targeting forkhead transcription factors FOXO3a and FOXM1 [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 73.

Abstract 3701: Mutant p53 cancers reprogram tumor-associated macrophages via exosomal miR-1246

Abstract TP53 mutants (mutp53) are involved in the pathogenesis of most human cancers. Specific mutp53 proteins gain oncogenic functions (GOF) distinct from the tumor suppressor activity of the wild-type protein. Tumor-associated-macrophages, a hallmark of solid tumors, are typically correlated with poor prognosis. Here we report a non-cell-autonomous mechanism whereby mutp53 cancer cells reprogram TAM to a tumor supportive and anti-inflammatory state. The colon cancer cells harboring GOF mutp53 selectively shed miR-1246-enriched exosomes. Uptake of these exosomes by neighboring macrophages triggers their miR-1246 dependent reprogramming into a cancer-promoting state. Mutp53-reprogammed TAM favor anti-inflammatory immunosuppression with increased activity of TGF-β. These findings, observed also in colon cancer patients, strongly support a microenvironmental GOF role for mutp53 in actively engaging the immune system to promote cancer progression and metastasis. Citation Format: Tomer Cooks, Ioannis S. Pateras, Lisa M. Jenkins, Keval M. Patel, Ana I. Robles, James Morris, Tim Forshew, Ettore Appella, Vassilis G. Gorgoulis, Curtis C. Harris. Mutant p53 cancers reprogram tumor-associated macrophages via exosomal miR-1246 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3701. doi:10.1158/1538-7445.AM2017-3701

Proteomic identification of ERP29 as a key chemoresistant factor activated by the aggregating p53 mutant Arg282Trp.

Mutation of the TP53 gene represents a prevalent genetic alteration in human cancers, and a subset of p53 mutants may form amyloid-like aggregates that contribute to the gain of oncogenic functions (GOFs) and chemoresistance. Here we identify the pathways that may mediate the aggregation-associated GOF by using combined proteomic analysis and genome-wide recruitment profiling. Mass spectrometry revealed activation of unfolded protein response (UPR) pathway and upregulation of endoplasmic reticulum protein 29 (ERp29) in R282WTP53-expressing cells that were exposed to cisplatin stress. Chromatin immunoprecipitation sequencing identified a significant 'CCCASS' binding motif of Arg282Trp, which is present in the promoter region of ERP29 gene. The mutant p53 upregulated ERP29 mRNA and protein expression levels, whereas targeting ERP29 by specific small interfering RNAs suppressed the chemoresistant effect of Arg282Trp. The anti-aggregation peptide ReACp53 significantly decreased ERP29 expression and suppressed the chemoresistant effect. These findings highlight a role of ERP29 in the acquired chemoresistance of cancer cells expressing the aggregating p53 mutant Arg282Trp. Our results also suggest that ERP29-mediated GOF can be targeted by the anti-aggregation peptide ReACp53.

Restoration of tumor suppression in vivo by systemic delivery of chemically-modified PTEN mRNA nanoparticles.

11582 Background: The onset and maintenance of cancer frequently involves gain of oncogenic function along with loss of tumor suppression. PTEN is a well-characterized tumor suppressor gene that is lost or mutated in many human cancers including ~50% of metastatic castration-resistant prostate cancer (mCRPC). Reintroduction of functional PTEN for mCRPC treatment has proven difficult. Methods: PTEN mRNA was synthesized by in vitro transcription method and modified with ARCA capping and enzymatic polyadenylation, and then substituted with Pseudo-UTP, 5’-Methyl-CTP. A robust self-assembly approach was employed to prepare PTEN mRNA nanoparticles (NPs) using cationic lipid-like compound G0-C14 and PLGA polymer coated with lipid-PEG shell. PTEN expression in tumors and PI3K-AKT pathway were confirmed by IHC and western blot, respectively. Apoptosis was checked by flow cytometry and Tunel assays. In vivo toxicity was studied by hematologic and histologic tests, and immune response. Results: We successfully restored PTEN mRNA to PTEN-null prostate cancer (PCa) cells via systemic delivery of mRNA NPs. These mRNA NPs are stable in serum, demonstrate minimal toxicity, and provide highly effective transfection in PCa cells (substantially higher HA-PTEN expression than plasmid PTEN transfection) and PCa xenograft tumors, leading to ~85% inhibition of tumor cell growth in vitro and in vivo. We also confirm mRNA NP-mediated systemic restoration of PTEN function in PTEN-null PCa and delineate its tumor suppression through inhibition of the PI3K-AKT pathway and enhancement of apoptosis. Conclusions: The work provides proof of principle for the systemic reintroduction of mRNA-based tumor suppressor genes to tumors in vivo. Because PTEN loss is frequent in late-stage PCa, this approach may have feasibility in this patient population. Considering the strong potential of mRNA therapy and the lack of systemic studies of in vivo mRNA transfection of tumors, this study sheds light on the useful application of NP-mediated mRNA delivery for validating tumor suppressors (e.g., PTEN) as a therapeutic target in cancer treatment where loss of a tumor suppressor contributes to the underlying genetic mechanism of cancer.

Chemoresistance of Cancer Cells: Oncogenic Mutation of the p53 Tumor Suppressor Gene

The mechanism for the chemoresistance of cancer cells is quite complicated. Most of the anti-cancer drugs are known to induce apoptosis in cancer cells, as well as in normal cells, in part by the activation of the p53 pathway. p53 is often referred to as the 'guardian of the genome' and is one of the most investigated tumor suppressor genes. A majority of human tumors harbor p53 mutations which can influence the effect of chemotherapy. Thus pre-therapeutic evaluation of the p53 gene can be extremely informative for patients with diverse malignancies. In this mini review, we introduce a p53 functional analysis system in mammalian cells, which can identify different types of p53 abnormalities, such as loss of function, dominant negative function, or gain of oncogenic function. The kinetic analysis of mutated-p53 may help to determine the therapeutic strategy in the individual patients with several malignancies. Furthermore, detailed information on the mutated p53 gene in cancer cells might provide useful suggestions for developing new anti-cancer drugs. Keywords: Chemoresistance, head and neck cancer, oncogenic mutation, p53, p53-target genes, tumor suppressor gene.

Oncogenic Mutant p53 Gain of Function Nourishes the Vicious Cycle of Tumor Development and Cancer Stem-Cell Formation.

More than half of human tumors harbor an inactivated p53 tumor-suppressor gene. It is well accepted that mutant p53 shows an oncogenic gain-of-function (GOF) activity that facilitates the transformed phenotype of cancer cells. In addition, a growing body of evidence supports the notion that cancer stem cells comprise a seminal constituent in the initiation and progression of cancer development. Here, we elaborate on the mutant p53 oncogenic GOF leading toward the acquisition of a transformed phenotype, as well as placing mutant p53 as a major component in the establishment of cancer stem cell entity. Therefore, therapy targeted toward cancer stem cells harboring mutant p53 is expected to pave the way to eradicate tumor growth and recurrence.

Gain-of-function miRNA signature by mutant p53 associates with poor cancer outcome.

Missense mutation of p53 not only impairs its tumor suppression function, but also causes oncogenic gain of function (GOF). The molecular underpinning of mutant p53 (mutp53) GOF is not fully understood, especially for the potential roles of non-coding genes. Here we identify the microRNA expression profile (microRNAome) of mutp53 on Arg282 by controlled microarray experiments, and clarify the prognostic significance of mutp53-regulated miRNAs in cancers. A predominant repression effect on miRNA expression was found for mutant p53, with 183 significantly downregulated and only 12 upregulated miRNAs. Mutp53 and wild-type (wtp53) commonly upregulate let-7i, and other two miRNAs were upregulated by wtp53 but repressed by mutp53 (miR-610 and miR-3065–3p). Based the mutp53-regulated miRNA signature, a non-negative matrix factorization (NMF) model classified gastric cancer (GC) cases into subgroups with significantly different Disease-free survival (Kaplan-Meier test, P = 0.013). In contrast, the NMF model based on all miRNAs did not associate with cancer outcome. The mutp53 miRNA signature associated with the outcomes of breast cancer (P = 0.024) and hepatocellular cancer (P = 0.012). The miRPath analysis revealed that mutp53-suppressed miRNAs associate with Hippo, TGF-β and stem cell signaling pathways. Taken together, our results highlight a miRNA-mediated GOF mechanism of mutant p53 on Arg282, and suggest the prognostic potential of mutp53-associated miRNA signature.

Abstract 34: Investigating PIN1 as a genetic modifier in Li-Fraumeni syndrome

Abstract Background: Li-Fraumeni syndrome (LFS) is characterized by marked clinical heterogeneity in site and age of cancer onset. The prolyl-isomerase, Pin1 (peptidylprolyl cis/trans isomerase, NIMA-interacting 1), has been shown to serve a fundamental role in mutant TP53 oncogenic gain of function and the promotion of aggressive cancer phenotypes. We sought to determine whether alterations in PIN1 modify the phenotype of patients with germline mutations in TP53. Methods: The PIN1 promoter and coding regions were sequenced from peripheral blood lymphocyte samples of 107 individuals with Li-Fraumeni syndrome and documented TP53 mutations. The expression and activity levels of PIN1 were determined in paired patient-derived lymphoblastoid cell lines. Results: Multivariate analysis of PIN1 variants with TP53 mutation status and selected clinical parameters is in progress. A comprehensive summary of observed associations will be presented. Conclusions: Demonstration of a modifying effect of PIN1 on the cancer phenotype conferred by a germline TP53 mutation may aid in the management of patients with Li-Fraumeni syndrome, including the implementation of patient-specific modifications of surveillance strategies for pre-symptomatic tumor detection. Citation Format: Anita Villani, Ana Novokmet, David Malkin. Investigating PIN1 as a genetic modifier in Li-Fraumeni syndrome. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr 34. doi:10.1158/1538-7445.CANSUSC14-34

Mutant p53 exerts oncogenic functions by modulating cancer cell metabolism

The metabolic function of p53 is important for its oncosuppressive function. Mutant p53 (mutp53) with gain of oncogenic function can regulate cell metabolism. Our recent study revealed a novel transcription-independent mechanism for a gain-of-function mutp53 that directly inhibits activation of adenosine monophosphate-activated protein kinase (AMPK) to promote cancer cell metabolism.

A mutant p53/let-7i-axis-regulated gene network drives cell migration, invasion and metastasis

Most p53 mutations in human cancers are missense mutations resulting in a full-length mutant p53 protein. Besides losing tumor suppressor activity, some hotspot p53 mutants gain oncogenic functions. This effect is mediated in part, through gene expression changes due to inhibition of p63 and p73 by mutant p53 at their target gene promoters. Here, we report that the tumor suppressor microRNA let-7i is downregulated by mutant p53 in multiple cell lines expressing endogenous mutant p53. In breast cancer patients, significantly decreased let-7i levels were associated with missense mutations in p53. Chromatin immunoprecipitation and promoter luciferase assays established let-7i as a transcriptional target of mutant p53 through p63. Introduction of let-7i to mutant p53 cells significantly inhibited migration, invasion and metastasis by repressing a network of oncogenes including E2F5, LIN28B, MYC and NRAS. Our findings demonstrate that repression of let-7i expression by mutant p53 has a key role in enhancing migration, invasion and metastasis.

SLC25A1, or CIC, is a novel transcriptional target of mutant p53 and a negative tumor prognostic marker

Mutations of the p53 gene hallmark many human cancers. Several p53 mutant proteins acquire the capability to promote cancer progression and metastasis, a phenomenon defined as Gain of Oncogenic Function (GOF). The downstream targets by which GOF p53 mutants perturb cellular programs relevant to oncogenesis are only partially known. We have previously demonstrated that SLC25A1 (CIC) promotes tumorigenesis, while its inhibition blunts tumor growth. We now report that CIC is a direct transcriptional target of several p53 mutants. We identify a novel interaction between mutant p53 (mutp53) and the transcription factor FOXO-1 which is responsible for regulation of CIC expression levels. Tumor cells harboring mutp53 display higher CIC levels relative to p53 null or wild-type tumors, and inhibition of CIC activity blunts mutp53-driven tumor growth, partially overcoming GOF activity. CIC inhibition also enhances the chemotherapeutic potential of platinum-based agents. Finally, we found that elevated CIC levels predict poor survival outcome in tumors hallmarked by high frequency of p53 mutations. Our results identify CIC as a novel target of mutp53 and imply that the employment of CIC inhibitors may improve survival rates and reduce chemo-resistance in tumors harboring these types of mutations, which are among the most intractable forms of cancers.

Disorders of the cMyb proto-oncogene expression and its significance in the course of ATL development

Accumulation of genetic disorders in HTLV-1 infected cells underlies ATL leukemogenesis, yet the actual genetic events responsible for cellular transformation have not been fully elucidated. Based on gene expression profiling in 52 ATL patients, 40 HTLV-1 carriers, and 21 healthy volunteers, we determined several potential risk-indicator genes of ATL, including cMyb. cMyb is the proto-oncogene of vMyb, the oncoprotein of avian myeloblastosis virus, governing hematopoietic cell differentiation. Required for differentiation of DN3, survival of DP, and generation of CD4+-SP cells, cMyb is not expressed at a detectable level in mature T-cells. Among well-known 7 isoforms, cMyb-9A and -10A, lacking the cis-acting negative regulatory domain (NRD) same as vMyb oncoprotein, are known to be molecules of “gain of oncogenic function”. We demonstrated that the mRNA levels of cmyb-9a and -10a were drastically elevated in ATL cells. Moreover, cMyb-9A protein was overexpressed in PBMC of HTLV-1 carriers and ATL patients. cMyb-9A showed the highest transactivation of HTLV-1 LTR, which is one of the cMyb targets, among 7 isoforms. The level of cMyb is known to be regulated by SUMOylation through the NRD. As expected, SUMOylation assay showed that cMyb-9A was not effectively SUMOylated, and its activity was not suppressed. Finally, cMyb-9A exhibited a significantly higher transforming activity than WT-cMyb. Upon confirming that cMyb-9A is released from the negative-regulatory circuit of cMyb, we speculate that overexpression of cMyb-9A in HTLV-1 infected cells has a strong link to disorders in cellular homeostasis by overruling its target gene expression, thus accelerating transformation process to ATL.