Inactivation In Human Cancer Research Articles

FBXW7 tumor suppressor regulation by dualspecificity tyrosine-regulated kinase 2

FBXW7 is a member of the F-box protein family, which functions as the substrate recognition component of the SCF E3 ubiquitin ligase. FBXW7 is a main tumor suppressor due to its ability to control proteasome-mediated degradation of several oncoproteins such as c-Jun, c-Myc, Cyclin E1, mTOR, and Notch1-IC. FBXW7 inactivation in human cancers results from a somatic mutation or downregulation of its protein levels. This work describes a novel regulatory mechanism for FBXW7 dependent on the serine/threonine protein kinase DYRK2. We show that DYRK2 interacts with and phosphorylates FBXW7 resulting in its proteasome-mediated degradation. DYRK2-dependent FBXW7 destabilization is independent of its ubiquitin ligase activity. The functional analysis demonstrates the existence of DYRK2-dependent regulatory mechanisms for key FBXW7 substrates. Finally, we provide evidence indicating that DYRK2-dependent regulation of FBXW7 protein accumulation contributes to cytotoxic effects in response to chemotherapy agents such as Doxorubicin or Paclitaxel in colorectal cancer cell lines and to BET inhibitors in T-cell acute lymphoblastic leukemia cell lines. Altogether, this work reveals a new regulatory axis, DYRK2/FBXW7, which provides an understanding of the role of these two proteins in tumor progression and DNA damage responses.

Unraveling the complexity: A comprehensive analysis of the PP2A in cancer and its potential for novel targeted therapies.

Protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase considered a potent tumor suppressor that critically regulates diverse cellular processes, including cell cycle progression, apoptosis, or DNA repair. PP2A is typically downregulated in cancers but mechanisms for its inactivation in human cancers are poorly understood. PP2A represents a family of more than 60 phosphatases. According to cellular context, each heterotrimeric PP2A holoenzyme exerts a unique role in cancer, and PP2A isoforms can act either as tumor suppressors or as promoters. Due to wide structural diversity, PP2A has been considered undruggable. However, increasing knowledge predisposes PP2A diversity to therapeutical targeting for the treatment of a broad range of cancer pathologies, including drug resistance or cloaking immune surveillance. In this review, we discuss the regulatory role of PP2A in cancer, its regulation by microRNA and hypoxia, its contribution to therapy resistance development, and the therapeutic potential of direct and indirect targeting, or combinatory administration with other anti-cancer drugs to improve cancer treatment outcomes.

TET deficiency perturbs mature B cell homeostasis and promotes oncogenesis associated with accumulation of G-quadruplex and R-loop structures.

Enzymes of the TET family are methylcytosine dioxygenases that undergo frequent mutational or functional inactivation in human cancers. Recurrent loss-of-function mutations in TET proteins are frequent in human Diffuse Large B-Cell Lymphoma (DLBCL). Here we investigate the role of TET proteins in B-cell homeostasis and development of B cell lymphomas with features of DLBCL. We show that deletion of Tet2 and Tet3 genes in mature B cells in mice perturbs B-cell homeostasis and results in spontaneous development of germinal center-derived B cell lymphomas with increased G-quadruplexes and R-loops. At a genome-wide level, G-quadruplexes and R-loops were associated with increased DNA double strand breaks at immunoglobulin switch regions. Deletion of the DNA methyltransferase DNMT1 in TET-deficient B cells prevented expansion of germinal center B cells, diminished the accumulation of G-quadruplexes and R-loops, and delayed B lymphoma development, consistent with the opposing functions of DNMT and TET enzymes in DNA methylation and demethylation. CRISPR-mediated depletion of nucleases and helicases that regulate G-quadruplexes and R-loops decreased the viability of TET-deficient B cells. Our studies suggest a molecular mechanism by which TET loss-of-function might predispose to development of B cell malignancies.

P53 deficiency triggers dysregulation of diverse cellular processes in physiological oxygen

The mechanisms by which TP53, the most frequently mutated gene in human cancer, suppresses tumorigenesis remain unclear. p53 modulates various cellular processes, such as apoptosis and proliferation, which has led to distinct cellular mechanisms being proposed for p53-mediated tumor suppression in different contexts. Here, we asked whether during tumor suppression p53 might instead regulate a wide range of cellular processes. Analysis of mouse and human oncogene-expressing wild-type and p53-deficient cells in physiological oxygen conditions revealed that p53 loss concurrently impacts numerous distinct cellular processes, including apoptosis, genome stabilization, DNA repair, metabolism, migration, and invasion. Notably, some phenotypes were uncovered only in physiological oxygen. Transcriptomic analysis in this setting highlighted underappreciated functions modulated by p53, including actin dynamics. Collectively, these results suggest that p53 simultaneously governs diverse cellular processes during transformation suppression, an aspect of p53 function that would provide a clear rationale for its frequent inactivation in human cancer.

Abstract 162: DNA methylation activates TP73 expression in human breast cancer tumorigenesis

Abstract Epigenetic events that cause changes in gene expression are common in human cancers. However, the connection between TP73 expression and its epigenetic functionality in tumorigenesis has not been widely explored. As a transcription factor, p73 is activated in a similar manner to p53 in response to DNA damage and regulates the expression of downstream genes involved in cell cycle arrest and apoptosis. However, there are other compounding functions of this gene that reflect its non-tumor-related characters, thus making it very difficult to assess its specific role in tumorigenesis, which results from an accumulation of mutational and epigenetic changes that alter normal cell growth and survival pathways. TP73 is the homologue of the master tumor suppressor TP53 involved in cellular responses to stress and development. The TP73 gene encodes two different proteins TAp73 and ΔNp73 and maps to a region on chromosome 1p36 that is frequently deleted in neuroblastoma and other tumors and is thought to contain multiple tumor suppressor genes. However, the analysis of p73-knockout mice yielded conflicting results with respect to tumor suppression. World-wide efforts in sequencing the TP73 gene in patient tumor samples have not provided evidence for genetic alterations as a common cause of TP73 inactivation in human cancers. Therefore, the role of TP73 in tumorigenesis has remained elusive to date. In this study, we determined TP73 expression in normal human mammary epithelial cells (HMEC), non-cancerous Li-Fraumeni Syndrome (LFS) skin fibroblasts, human breast cancer cell lines (MCF7 & MDA321), neuroblastoma cell lines (IMR32 & SK-N-SH) and normal skin fibroblast cell line (HS27). We also conducted an immunohistochemistry analysis of TP73 expression in human normal breast tissues, cancerous breast tissues and non-cancerous (cancer-adjacent) breast tissues. The results show that TP73 expression is evident only in cancer cell lines and breast cancer tissues. Moreover, methylation-specific PCR and bisulfite sequencing studies revealed that TP73 promoter is activated only in cancer cell lines by DNA methylation. Furthermore, ChIP assay results demonstrated that CTCF (a chromosomal networking protein CCCTC binding factor) and tumor protein p53 (TP53) bind to TP73 promoter and regulate TP73 expression. These observations demonstrate the existence of a positive correlation at the promoter site of TP73 between its expression and DNA methylation. However, TP73 expression is not involved in cell proliferation, which may prove significant for the development of future diagnostic and therapeutic applications. Citation Format: Zhixing Yao, David R. Yao, William Yu, Zaki A. Sherif. DNA methylation activates TP73 expression in human breast cancer tumorigenesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 162.

Abstract 4321: Epigenetic regulation of TP73 expression in HCC and GI cancer

Abstract Tumorigenesis results from an accumulation of mutational and epigenetic changes that alter normal cell growth and survival pathways. TP73 is the homologue of the master tumor suppressor TP53 involved in cellular responses to stress and development. The TP73 gene encodes two different protein isoforms, TAp73 and ΔNp73, and maps to a region on chromosome 1p36 that is frequently deleted in neuroblastoma and other tumors, and is thought to contain multiple tumor suppressor genes. However, the analysis of p73-knockout mice yielded conflicting results with respect to tumor suppression. World-wide efforts in sequencing the TP73 gene in patient tumor samples have not provided evidence for genetic alterations as a common cause of TP73 inactivation in human cancer. The role of TP73 in tumorigenesis has remained elusive to date. In this study, we isolated two stem cell lines from normal young and old human liver tissues to determine TP73 expression in normal human liver stem cell and hepatocellular cancer (HCC) cell lines (HepG2, SNU398, SNU449 & SNU475), gastrointestinal cancer (GI) cell lines (Caco2 & HCT116) and a normal skin fibroblast cell line (HS27). The results show that TP73 only expresses in cancer cell lines. Further immunohistochemistry studies on human normal liver tissues as well as adjacent normal and cancerous liver tissues from HCC patients confirm that TP73 only expresses in the cancerous tissues. Moreover, methylation-specific PCR and bisulfite sequencing studies revealed that TP73 promoter is activated only in cancer cell lines by DNA methylation. Furthermore, ChIP assay results demonstrated that CTCF (a chromosomal networking protein CCCTC binding factor) binds to TP73 promoter and regulates TP73 expression. Our observation may prove significant for the development of future therapeutic and diagnostic applications. Citation Format: Zhixing Yao, Cristina Di Poto, Habtom W. Ressom, Zaki A. Sherif. Epigenetic regulation of TP73 expression in HCC and GI cancer [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 4321.

Interferon regulatory factor 1 inactivation in human cancer.

Interferon regulatory factors (IRFs) are a group of closely related proteins collectively referred to as the IRF family. Members of this family were originally recognized for their roles in inflammatory responses; however, recent research has suggested that they are also involved in tumor biology. This review focusses on current knowledge of the roles of IRF-1 and IRF-2 in human cancer, with particular attention paid to the impact of IRF-1 inactivation. The different mechanisms underlying IRF-1 inactivation and their implications for human cancers and the potential importance of IRF-1 in immunotherapy are also summarized.

Abstract 2576: Benzyl isothiocyanate potentiates p53 signaling and antitumor effects against breast cancer through activation of p53-LKB1 and p73-LKB1 axes

Abstract Functional reactivation of p53 pathway, although arduous, can potentially provide a broad-based strategy for cancer therapy owing to frequent p53 inactivation in human cancer. Using a phosphoprotein-screening array, we found that Benzyl Isothiocynate, (BITC) increases p53 phosphorylation in breast cancer cells and reveal an important role of ERK and PRAS40/MDM2 in BITC-mediated p53 activation. We show that BITC rescues and activates p53-signaling network and inhibits growth of p53-mutant cells. Mechanistically, BITC induces p73 expression in p53-mutant cells, disrupts the interaction of p73 and mutant-p53, thereby releasing p73 from sequestration and allowing it to be transcriptionally active. Furthermore, BITC-induced p53 and p73 axes converge on tumor-suppressor LKB1 which is transcriptionally upregulated by p53 and p73 in p53-wild-type and p53-mutant cells respectively; and in a feed-forward mechanism, LKB1 tethers with p53 and p73 to get recruited to p53-responsive promoters. Analyses of BITC-treated xenografts using LKB1-null cells corroborate in vitro mechanistic findings and establish LKB1 as the key node whereby BITC potentiates as well as rescues p53-pathway in p53-wild-type as well as p53-mutant cells. These data provide first in vitro and in vivo evidence of the integral role of previously unrecognized crosstalk between BITC, p53/LKB1 and p73/LKB1 axes in breast tumor growth-inhibition. Citation Format: Arumugam Nagalingam, Bei Xie, Panjamurthy Kuppusamy, Nethaji Muniraj, Peter Langford, Balázs Győrffy, Neeraj Saxena, Dipali Sharma. Benzyl isothiocyanate potentiates p53 signaling and antitumor effects against breast cancer through activation of p53-LKB1 and p73-LKB1 axes [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 2576. doi:10.1158/1538-7445.AM2017-2576

Abstract 2573: TP73's regulation and expression in human cancer cell lines

Abstract TP73 is the homologue of the master tumor suppressor TP53. It is involved in cellular responses to stress and development. The TP73 gene encodes two different proteins, TAp73 and ΔNp73 and maps to a region on chromosome 1p36 that is frequently deleted in neuroblastoma and other tumors, and thought to contain multiple tumor suppressor genes. However, the analysis of p73-knockout mice yielded conflicting results with respect to tumor suppression. World-wide efforts in sequencing the TP73 gene in patient tumor samples have not provided evidence for genetic alterations as a common cause of p73 inactivation in human cancer. The role of TP73 in tumorigenesis has remained elusive to date. In this study, we isolated two stem cell lines from normal young and old human liver tissues and determined TP73 expression in human breast cancer cell lines (MCF7 & MDA321), human hepatocellular carcinoma (HCC) cell lines (HepG2, SNU398, SNU449 & SNU475), neuroblastoma cell lines (IMR32 & SK-N-SH), normal liver stem cell, non-cancerous Li-Fraumeni Syndrome (LFS) skin fibroblasts cells and normal skin fibroblasts. Results show that TAp73 only expresses in cancer cell lines. Moreover, when HepG2 and MDA231 were treated with paclitaxel, a cytoskeletal drug that targets tubulin, TAp73 expression was drastically down-regulated in HepG2 cells and abrogated in MDA231 cells at 1 to 5 µmol/L concentrations. Furthermore, ChIP assay results demonstrated that TP53 and CTCF (a chromosomal networking protein CCCTC binding factor) bind to TP73 promoter and regulate TP73 expression. Our observation may prove significant for the development of future therapeutic and diagnostic applications. *These authors contributed equally to this work. Citation Format: Zhixing Yao, David R. Yao, William Yu, Hassan Brim, Hassan Ashktorab, Zaki A. Sherif. TP73's regulation and expression in human cancer cell lines [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 2573. doi:10.1158/1538-7445.AM2017-2573

Loss of LZAP inactivates p53 and regulates sensitivity of cells to DNA damage in a p53-dependent manner

Chemotherapy and radiation, the two most common cancer therapies, exert their anticancer effects by causing damage to cellular DNA. However, systemic treatment damages DNA not only in cancer, but also in healthy cells, resulting in the progression of serious side effects and limiting efficacy of the treatment. Interestingly, in response to DNA damage, p53 seems to play an opposite role in normal and in the majority of cancer cells—wild-type p53 mediates apoptosis in healthy tissues, attributing to the side effects, whereas mutant p53 often is responsible for acquired cancer resistance to the treatment. Here, we show that leucine zipper-containing ARF-binding protein (LZAP) binds and stabilizes p53. LZAP depletion eliminates p53 protein independently of its mutation status, subsequently protecting wild-type p53 cells from DNA damage-induced cell death, while rendering cells expressing mutant p53 more sensitive to the treatment. In human non-small-cell lung cancer, LZAP levels correlated with p53 levels, suggesting that loss of LZAP may represent a novel mechanism of p53 inactivation in human cancer. Our studies establish LZAP as a p53 regulator and p53-dependent determinative of cell fate in response to DNA damaging treatment.

Benzyl Isothiocyanate potentiates p53 signaling and antitumor effects against breast cancer through activation of p53-LKB1 and p73-LKB1 axes

Functional reactivation of p53 pathway, although arduous, can potentially provide a broad-based strategy for cancer therapy owing to frequent p53 inactivation in human cancer. Using a phosphoprotein-screening array, we found that Benzyl Isothiocynate, (BITC) increases p53 phosphorylation in breast cancer cells and reveal an important role of ERK and PRAS40/MDM2 in BITC-mediated p53 activation. We show that BITC rescues and activates p53-signaling network and inhibits growth of p53-mutant cells. Mechanistically, BITC induces p73 expression in p53-mutant cells, disrupts the interaction of p73 and mutant-p53, thereby releasing p73 from sequestration and allowing it to be transcriptionally active. Furthermore, BITC-induced p53 and p73 axes converge on tumor-suppressor LKB1 which is transcriptionally upregulated by p53 and p73 in p53-wild-type and p53-mutant cells respectively; and in a feed-forward mechanism, LKB1 tethers with p53 and p73 to get recruited to p53-responsive promoters. Analyses of BITC-treated xenografts using LKB1-null cells corroborate in vitro mechanistic findings and establish LKB1 as the key node whereby BITC potentiates as well as rescues p53-pathway in p53-wild-type as well as p53-mutant cells. These data provide first in vitro and in vivo evidence of the integral role of previously unrecognized crosstalk between BITC, p53/LKB1 and p73/LKB1 axes in breast tumor growth-inhibition.

Transflip mutations produce deletions in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is driven by the inactivation of the tumor suppressor genes (TSGs), CDKN2A (P16) and SMAD4 (DPC4), commonly by homozygous deletions (HDs). Using a combination of high density single-nucleotide polymorphism (SNP) microarray and whole genome sequencing (WGS), we fine-mapped novel breakpoints surrounding deletions of CDKN2A and SMAD4 and characterized them by their underlying structural variants (SVs). Only one third of CDKN2A and SMAD4 deletions (6 of 18) were simple interstitial deletions, rather, the majority of deletions were caused by complex rearrangements, specifically, a translocation on one side of the TSG in combination with an inversion on the other side. We designate these as "TransFlip" mutations. Characteristics of TransFlip mutations are: (1) a propensity to target the TSGs CDKN2A and SMAD4 (P < 0.005), (2) not present in the germline of the examined samples, (3) non-recurrent breakpoints, (4) relatively small (47 bp to 3.4 kb) inversions, (5) inversions can be either telomeric or centromeric to the TSG, and (6) non-reciprocal, and non-recurrent translocations. TransFlip mutations are novel complex genomic rearrangements with unique breakpoint signatures in pancreatic cancer. We hypothesize that they are a common but poorly understood mechanism of TSG inactivation in human cancer. © 2015 Wiley Periodicals, Inc.

Targeting Tumor Suppressor p53 for Cancer Therapy: Strategies, Challenges and Opportunities

p53 is one of the most important tumor suppressor genes that is frequently mutated in human cancers. Generally, p53 functions as a transcription factor that is stabilized and activated by various genotoxic and cellular stress signals, such as DNA damage, hypoxia, oncogene activation and nutrient deprivation, consequently leading to cell cycle arrest, apoptosis, senescence and metabolic adaptation. p53 not only becomes functionally deficient in most cancers, but not infrequently mutant p53 also acquires dominant negative activity and oncogenic properties. p53 has remained an attractive target for cancer therapy. Strategies targeting p53 have been developed including gene therapy to restore p53 function, inhibition of p53-MDM2 interaction, restoration of mutant p53 to wild-type p53, targeting p53 family proteins, eliminating mutant p53, as well as p53-based vaccines. Some of these p53-targeted therapies have entered clinical trials. We discuss the therapeutic potential of p53, with particular focus on the therapeutic strategies to rescue p53 inactivation in human cancers. In addition, we discuss the challenges of p53-targeted therapy and new opportunities for the future.

Cancerous Inhibitor of Protein Phosphatase 2A, an Emerging Human Oncoprotein and a Potential Cancer Therapy Target

Protein phosphatase 2A (PP2A) complexes function as tumor suppressors by inhibiting the activity of several critical oncogenic signaling pathways. Consequently, inhibition of the PP2A phosphatase activity is one of many prerequisites for the transformation of normal human cells into cancerous cells. However, mechanisms for PP2A inactivation in human cancers are poorly understood. The aberrant expression of cancerous inhibitor of protein phosphatase 2A (CIP2A), a recently identified endogenous PP2A inhibitor in malignant cells, is one such mechanism. Various independent studies have validated CIP2A's role in promoting tumor growth and resistance to apoptosis and senescence-inducing therapies. Notably, high CIP2A expression predicts poor patient prognosis in several human cancer types. Among the oncogenic proteins dephosphorylated by PP2A, the MYC oncoprotein, which is phosphorylated at serine 62, has surfaced as a marker for the oncogenic activity of CIP2A. The positive-feedback loop between CIP2A and MYC augments the activity of MYC in cancer cells. In addition, CIP2A promotes the phosphorylation and activity of additional oncoproteins, including E2F1 and AKT. However, CIP2A is not essential for normal mouse growth and development. These findings indicate that CIP2A is a novel anticancer target based on PP2A reactivation and inhibition of the oncogenic activity of its downstream effectors. The potential approaches and feasibility of targeting CIP2A are discussed here.

Abstract 1587: Loss of LZAP inactivates p53 in head and neck cancer and regulates sensitivity of cells to DNA damage in the p53-dependent manner.

Abstract We previously reported that tumor suppressor LZAP is lost in a portion of head and neck squamous cell carcinomas and that LZAP inhibits NF-kB, while activating p53 in ARF-dependent and -independent fashions. The ability of LZAP to regulate p53 in the absence of ARF has been largely unexplored. Here, we show that LZAP depletion diminished expression of mutant and wild-type p53 protein. LZAP activity toward p53 was independent of ARF and the known p53 regulators, Wip1 and HDM2. Loss of LZAP decreased p53 translation and destabilized p53 protein due, at least partially, to increased nucleolin expression. Knockdown of LZAP abrogated p53 stabilization induced by ionizing radiation, and LZAP depletion protected wild-type p53 cells from radiation or chemotherapeutic agents, while rendering cells expressing mutant or no p53 more sensitive. In human HNSCC, LZAP levels correlated with p53 levels, and remarkably, tumors with low LZ were less likely to have p53 mutations. These data suggest that loss of LZAP represents a novel mechanism of p53 inactivation in human cancer. Given these findings, inhibition of LZAP activity toward p53 for patients with tumors harboring p53 mutations may represent a therapeutic strategy to simultaneously sensitize tumors while protecting normal tissues from radiation or chemotherapy. Citation Format: Dan Liu, Natalia Issaeva, Wendell G. Yarbrough. Loss of LZAP inactivates p53 in head and neck cancer and regulates sensitivity of cells to DNA damage in the p53-dependent manner. [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 1587. doi:10.1158/1538-7445.AM2013-1587

CpG island hypermethylation-associated silencing of small nucleolar RNAs in human cancer

Much effort in cancer research has focused on the tiny part of our genome that codes for mRNA. However, it has recently been recognized that microRNAs also contribute decisively to tumorigenesis. Studies have also shown that epigenetic silencing by CpG island hypermethylation of microRNAs with tumor suppressor activities is a common feature of human cancer. The importance of other classes of non-coding RNAs, such as long intergenic ncRNAs (lincRNAs) and transcribed ultraconserved regions (T-UCRs) as altered elements in neoplasia, is also gaining recognition. Thus, we wondered whether there were other ncRNAs undergoing CpG island hypermethylation-associated inactivation in cancer cells. We focused on the small nucleolar RNAs (snoRNAs), a subset of ncRNA with a wide variety of cellular functions, such as chemical modification of RNA, pre-RNA processing and control of alternative splicing. By data mining snoRNA databases and the scientific literature, we selected 49 snoRNAs that had a CpG island within ≤ 2 Kb or that were processed from a host gene with a 5′-CpG island. Bisulfite genomic sequencing of multiple clones in normal colon mucosa and the colorectal cancer cell line HCT-116 showed that 46 snoRNAs were equally methylated in the two samples: completely unmethylated (n = 26) or fully methylated (n = 20). Most interestingly, the host gene-associated 5′-CpG islands of the snoRNAs SNORD123, U70C and ACA59B were hypermethylated in the cancer cells but not in the corresponding normal tissue. CpG island hypermethylation was associated with the transcriptional silencing of the respective snoRNAs. Results of a DNA methylation microarray platform in a comprehensive collection of normal tissues, cancer cell lines and primary malignancies demonstrated that the observed hypermethylation of snoRNAs was a common feature of various tumor types, particularly in leukemias. Overall, our findings indicate the existence of a new subclass of ncRNAs, snoRNAs, that are targeted by epigenetic inactivation in human cancer.

RUNX3 interactome reveals novel centrosomal targeting of RUNX family of transcription factors

RUNX family proteins are critical regulators of lineage differentiation during development. The high prevalence of RUNX mutation/epigenetic inactivation in human cancer indicates a causative role for dysfunctional RUNX in carcinogenesis. This is supported by well-documented evidence of functional interaction of RUNX with components of major oncogenic or tumor suppressive signaling pathways such as TGFβ and Wnt. Here, we explore the binding partners of RUNX3 proteins to further define the scope of RUNX3 function. Using a mass spectrometry-based approach, we found that RUNX3 binds to centrosomal protein rootletin. This led us to uncover the presence of RUNX proteins at the centrosome. Our findings suggest a potential function for RUNX3 during mitosis.

Abstract 1172: Prodigiosin and its structural analogue rescue deficient p53 pathway signaling and anti-tumor effects via p73 upregulation in p53 mutant and null colorectal cancer cells

Abstract The tumor suppressor p53 gene is mutated in over 50% of human cancers, and this is associated with resistance to radiotherapy and chemotherapy as well as tumor progression. Therefore, p53 pathway functional reactivation in p53-deficient cancers is an attractive strategy for cancer therapy and may address an unmet need in the clinic for progressive therapy-refractory human tumors. We previously showed that Prodigiosin and its structure analogue can reactivate the p53 pathway in p53 mutant and null colorectal cancer cells (Hong et al. AACR 2011, abstract 607). Here, we examined the mechanism of p53 pathway reactivation by Prodigiosin and its structural analogue in colorectal cancer cells. p73, a p53 family protein, shares a high degree of sequence homology with p53 and regulates the expression of similar genes by binding to p53 responsive elements within gene promoters and performs similar functions to p53. However, unlike p53, p73 is not a target for mutational inactivation in human cancer. In the study, we hypothesized that Prodigiosin and its structural analogue induce p73 expression to activate the p53 pathway and further induce cellular apoptosis in p53-deficient cancer cells. We showed that Prodigiosin and its structural analogue induced p73 expression in p53 mutant SW480 and p53−/− HCT116 cells. We also showed that the induction of p53-responsive transcriptional activities by Prodigiosin and its structural analogue was reduced in SW480 cells with p73 stable knockdown, as compared with parental SW480 cells. Western blot analysis indicated that the induction of a p53 target protein DR5 by Prodigiosin and its structural analogue was decreased in SW480 cells with p73 stable knockdown, as compared with parental SW480 cells. The sub-G1 analysis demonstrated that Prodigiosin and its structural analogue induced less apoptosis in SW480 cells with p73 stable knockdown than parental SW480 cells. Together, these findings indicate that p73 upregulation is involved in the mechanism of p53 pathway activation and apoptotic induction by Prodigiosin and its structural analogue. The study suggests that p73 induction may be a useful alternative way to reactivate p53 pathway in p53 mutant and null cancer cells and provides important mechanistic insights into the activity of a potent p53 pathway function restoring small molecule that is a candidate therapeutic for human cancer. This work was supported by the NCI Developmental Therapeutics Program through NCI Contract N01-CN-43302. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1172. doi:1538-7445.AM2012-1172

A Frameshift Mutation of the Pro-Apoptotic VDAC1 Gene in Cancers with Microsatellite Instability

A Frameshift Mutation of the Pro-Apoptotic VDAC1 Gene in Cancers with Microsatellite Instability

Silencing of SHP1 Is Frequent in Phospho-STAT3 Positive Diffuse Large Cell Lymphoma and This Silencing Is Independent of Methylation of Cpg Islands,

Abstract 3678The JAK/STAT pathway is becoming a relevant target for lymphoma therapy and inhibitors of the pathway are now in clinical trial. It would be ideal to identify patients for trial by assessment of pathway activation. We analyzed the primary tumors from 50 patients with diffuse large B cell lymphoma (DLBCL) using antibody against phosphorylated tyrosine signal transducer and activator of transcription 3 (STAT3) revealed that 52% (21/40) of DLBCL tumors express tyrosine phosphorylated STAT3 (pSTAT3). There are several potential mechanisms of activation of the JAK/STAT pathway. Protein tyrosine phosphatases such as SHP1 are key negative regulators of this pathway. SHP-1 is also considered a tumor suppressor gene because of its ability to antagonize the action of tyrosine kinases. Persistent activation of STAT3 seems to play a critical role in the pathogenesis of several malignancies. In an attempt to determine mechanism of STAT3 phosphorylation, we analyzed SHP1 expression and its correlation to STAT3 phosphorylation in DLBCL tumors.We analyzed SHP1 expression by immunohistochemistry (IHC) in a tissue microarray composed of 27 DLBCL tumor samples. SHP1 was found silenced in 33% (7/27) of DLBCL cases. To confirm our IHC data SHP1 mRNA expression was also analyzed in DLBCL patients (n=7) and CD19+ normal B cells (n=3) using reverse transcriptase polymerase chain reaction (RT-PCR). SHP1 expression was found silenced in 57% (4/7) of cases, however normal B cells expressed a significant amount of SHP1 mRNA transcripts. We next sought to determine the mechanism of SHP1 silencing in these patients. DNA methylation is the most common mechanism of gene silencing and gene inactivation in human cancers and methylation of a cluster of CPG sites near the proximal SHP1 promoter 2 has been shown in myeloma and T cell lymphoma cells. We analyzed the frequency of SHP1 DNA methylation in 40 DLBCL patients and in DLBCL cell lines by analyzing the proximal promoter regions for SHP1 with methylation specific (MSP) and unmethylation specific (UMSP) PCR primers. We found SHP1 methylation in 2.5% (1/40) DLBCL tumors and none of the DLBCL cell lines tested (OCILy3, OCILy10, SUDHL2, SUDHL6, OCILy19 and OCILy1) were positive for methylation. Furthermore, bisulphite sequencing of the MSP product confirmed methylated cytosines in the one MSP-positive DLBCL patient sample and in the positive control (methylated DNA). We next confirmed our MSP methylation results in the 40 DLBCL tumors DNA with a high resolution melting (HRM) methylation array for accurate detection of changes in the CpG methylation. The primers were designed to flank CpG islands, not to specifically amplify (converted) methylated or unmethylated DNA (as in MSP PCR). Data were normalized to an unmethylated control. Based on the melting profile, there was no methylation in the controls but detectable methylation (>5%) in the SHP1 promoter region was found in 10% (4/40) of DLBCL samples. The one sample that was positive with MSP PCR was also >50% methylated by HRM. Next we analyzed whether there is any correlation between SHP1 expression and tyrosine phoshorylated STAT3 (pSTAT3). In the 27 cases of DLBCL studied, 16 were pSTAT3 negative and 11 were pSTAT3 positive by IHC. 50% (8/16) of the pSTAT3 negative cases had very high expression of SHP1. Similarly, 45 % (5/11) of pSTAT3 positive cases were SHP1 negative consistent with loss of SHP1 gene expression. These data suggest that loss of SHP1 expression is one of several potential mechanisms that can contribute to increased tyrosine STAT3 activation in DLBCL. In addition, these data suggest that CPG island promoter hypermethylation in the SHP1 promoter is not associated with SHP1 gene silencing in DLBCL tumors. Disclosures:No relevant conflicts of interest to declare.