Cytoplasmic Localization Of P53 Research Articles

A role of cytoplasmic p53 in the regulation of metabolism shown by bat-mimicking p53 NLS mutant mice

The transcription factor p53 suppresses tumorigenesis via a wide-ranging, concerted set of functions. Although several studies have identified cytoplasmic, transcription-independent functions of p53, the biological relevance of these activities has not been fully elucidated, particularly invivo. Here, we generated a mouse model with a p53K316P mutation, which mimics a naturally occurring p53 nuclear localization signal (NLS) change observed in bat species. We find that the p53K316P mutation increases cytoplasmic localization of p53 and promotes a pleiotropic metabolic phenotype that includes increased adiposity, increased de novo lipogenesis, and decreased lactate generation. Mechanistic studies show that, independent of its transactivation function, p53K316P interacts with lactate dehydrogenase B (LDHB) and alters the composition and enzymatic activities of LDH complex favoring pyruvate generation and hindering lactate production. Overall, the study identifies a role for cytoplasmic p53 in the regulation of metabolism that favors energy generation and storage.

Organophosphorus Flame Retardant TDCPP Displays Genotoxic and Carcinogenic Risks in Human Liver Cells.

Tris(1,3-Dichloro-2-propyl)phosphate (TDCPP) is an organophosphorus flame retardant (OPFR) widely used in a variety of consumer products (plastics, furniture, paints, foams, and electronics). Scientific evidence has affirmed the toxicological effects of TDCPP in in vitro and in vivo test models; however, its genotoxicity and carcinogenic effects in human cells are still obscure. Herein, we present genotoxic and carcinogenic properties of TDCPP in human liver cells (HepG2). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and neutral red uptake (NRU) assays demonstrated survival reduction in HepG2 cells after 3 days of exposure at higher concentrations (100–400 μM) of TDCPP. Comet assay and flow cytometric cell cycle experiments showed DNA damage and apoptosis in HepG2 cells after 3 days of TDCPP exposure. TDCPP treatment incremented the intracellular reactive oxygen species (ROS), nitric oxide (NO), Ca2+ influx, and esterase level in exposed cells. HepG2 mitochondrial membrane potential (ΔΨm) significantly declined and cytoplasmic localization of P53, caspase 3, and caspase 9 increased after TDCPP exposure. qPCR array quantification of the human cancer pathway revealed the upregulation of 11 genes and downregulation of two genes in TDCPP-exposed HepG2 cells. Overall, this is the first study to explicitly validate the fact that TDCPP bears the genotoxic, hepatotoxic, and carcinogenic potential, which may jeopardize human health.

Abstract 3470: The antagonistic functional duality of p21WAF1/CIP1 in cells derived from multicellular tumor spheroids

Abstract The cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21) is a key mediator of p53-dependent cell cycle arrest after DNA damage, in addition to p53-independent mechanisms. Being one of the major transcriptional targets of p53 and due to its anti-cell proliferative activity, p21 was considered initially as a potent tumor suppressor. However, emerging studies now show the anti-apoptotic and pro-cell proliferative effects of p21, highlighting the oncogenic role of p21 in cancer. In particular, p21 results in genomic instability and the development of aggressive and chemo-resistant traits in a subset of highly proliferating tumor cells through p53-independent pathways. The cellular localization of p21 has been proposed to be critical either in promoting cell survival or in inhibiting cell growth. Herein, using a combination of gene knockout, cytotoxicity assay, immunofluorescence, immunoprecipitation, and mass spectrometry techniques, we study the consequence of nuclear and cytoplasmic localization of p21 on cell survival and drug response in cells derived from multicellular spheroids of multiple human cancer cell lines. Our study shows that cells derived from multicellular spheroids show an increased induction of p21 despite the reversal from three-dimensional to two-dimensional cultures. The p21-overexpressing, highly proliferative cells are significantly resistant to a number of standard anti-cancer agents. Further analysis shows that the majority of p21 in this subset of spheroid-derived cells is localized in the cytoplasm and forms a potential 'anti-apoptosome'-like complex with mitochondrial apoptosis-associated proteins. These findings add to the shifting paradigm on the oncogenic role p21 due to cellular mislocalization in tumor cells. Citation Format: Viswanath Das, Narendran Annadurai, Dušan Holub, Marián Hajdúch. The antagonistic functional duality of p21WAF1/CIP1 in cells derived from multicellular tumor spheroids [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 3470.

Prevention of Wogonin on Colorectal Cancer Tumorigenesis by Regulating p53 Nuclear Translocation.

The tumor suppressor protein p53 plays an important role in the development and progression of colon cancer, and the subcellular organelle localization directly affects its function. Wogonin (5,7-dihydroxy-8-methoxyflavone), a mono-flavonoid extracted from root of Scutellaria baicalensis Georgi, possesses acceptable toxicity and has been used in colorectal cancer (CRC) chemoprevention in pre-clinical trials by oncologist. However, the underlying anti-colon cancer mechanisms of wogonin are not yet fully understood. In the present study, the effect of wogonin on the initiation and development of colitis-associated cancer through p53 nuclear translocation was explored. AOM-DSS CRC animal model and human CRC HCT-116 cell model were used to evaluate the in vivo and in vitro anti-colon cancer action of wogonin. We observed that wogonin showed a dramaticlly preventive effect on colon cancer. Our results showed that wogonin caused apoptotic cell death in human CRC HCT-116 cell through increased endoplasmic reticulum (ER) stress. Meanwhile, excessive ER stress facilitated the cytoplasmic localization of p53 through increasing phosphor-p53 at S315 and S376 sites, induced caspase-dependent apoptosis and inhibited autophagy. Furthermore, we verified the chemoprevention effect and toxicity of wogonin in vivo by utilizing an AOM-DSS colon cancer animal model. We found that wogonin not only reduced tumor multiplicity, preserved colon length to normal (6.79 ± 0.34 to 7.41 ± 0.56, P < 0.05) but also didn’t induce side effects on various organs. In conclusion, these results explain the anti-tumor effect of wogonin in CRC and suggest wogonin as a potential therapeutic candidate for the therapeutic strategy in CRC treatment.

Abstract 2396: Short-term calorie restriction alters expression of tumor suppressor p21 in HER2-overexpressing breast cancer

Abstract Breast cancer (BC) is the most common noncutaneous cancer among US women, and ~20% of these cancers overproduce the growth-promoting tyrosine kinase receptor, HER2. Although there are targeted treatments available for HER2-overexpressing BC, there remains an urgent need to identify new treatment strategies since this disease is associated with poor prognosis, resistance to therapy, and high risk of recurrence. We have established that calorie restriction (CR; 30% reduction in total energy relative to ad libitum-fed controls) has significant tumor suppressive effects across all breast cancer subtypes and has potential to be utilized as an adjunctive therapy. This study investigates the mechanisms by which CR decreases HER2-overexpressing BC progression. To mimic CR in vitro, murine MMTV-neu cells that overexpress neu, the rodent homolog of HER2, were treated with media containing reduced serum (1%), reduced glucose (1mM), or reduced serum and reduced glucose (1%/1mM) compared to control media (10% serum, 25mM glucose). MTT assays demonstrated that both serum restricted medias (1% and 1%/1mM) significantly decreased cellular viability (p&amp;lt;0.05) compared to control. To investigate the mechanism of this alteration, western blotting analysis of proteins associated with HER2 BC progression was conducted. In MMTV-neu cells treated with serum restricted medias (1% and 1%/1mM), there was a significant decrease in activity of tumor suppressor protein p21. Both p21 and phospho-21 expression were decreased with serum restriction, as well as the ratio of phospho-p21 relative to p21 (p&amp;lt;0.05). Phosphorylated p21 is localized to the cytoplasm and allows BC cells to evade apoptosis and proliferate uncontrollably. These results suggest that short-term CR, achieved specifically through serum restriction alone or in combination with glucose restriction, is associated with decreased phosphorylation and cytoplasmic localization of p21, which may be responsible for modulating anti-proliferative activities. This finding is significant because clinical studies have found that increased cytoplasmic p21 in HER2-overexpressing BC predicted reduced survival in patients at 5 years. Utilizing CR or pharmacologic regimens that mimic CR in conjunction with existing therapies may prevent or reverse cytoplasmic p21 localization in this cancer subtype, highlighting the importance of this investigation. Studies are ongoing in MMTV-neu cells transfected with plasmids encoding mutated p21 proteins that are constitutively expressed in either the cytoplasm or the nucleus. This will confirm the extent to which localization of p21 is responsible for CR-induced alterations in HER2-overexpressing BC cell proliferation. Citation Format: Magdalena A. Rainey, Laura A. Smith, Ciara H. O'Flanagan, Stephen D. Hursting. Short-term calorie restriction alters expression of tumor suppressor p21 in HER2-overexpressing breast cancer [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 2396.

Androgen induces G3BP2 and SUMO-mediated p53 nuclear export in prostate cancer.

The androgen receptor (AR) has a central role in prostate cancer progression, particularly treatment-resistance disease including castration-resistant prostate cancer. Loss of the p53 tumor suppressor, a nuclear transcription factor, is also known to contribute to prostate malignancy. Here we report that p53 is translocated to the cytoplasm by androgen-mediated induction of G3BP2, a newly described direct target gene of AR. G3BP2 induces both cell cycle progression and blocks apoptosis. Translocation of p53 is regulated by androgen-dependent sumoylation mediated by the G3BP2-interacting SUMO-E3 ligase, RanBP2. G3BP2 knockdown results in reduced tumor growth and increased nuclear p53 accumulation in mouse xenograft models of prostate cancer with or without long-term androgen deprivation. Moreover, strong cytoplasmic p53 localization is correlated clinically with elevated G3BP2 expression and predicts poor prognosis and disease progression to the hormone-refractory state. Our findings reveal a new AR-mediated mechanism of p53 inhibition that promotes treatment-resistant prostate cancer.

ER chaperone GRP78 regulates autophagy by modulation of p53 localization.

GRP78 (glucose regulated protein 78) is a major Endoplasmic Reticulum (ER) chaperone that plays a pivotal role in normal ER functioning. Its increased expression also works as an indicator of ER stress. Its anti-apoptotic and pro-autophagic activity makes it an intriguing target to study the relationship between GRP78 and p53, which is also a major regulator of apoptosis and autophagy. Here, we studied the effect of Rotenone and Parathion on human lung cancer cells (A549 cell line) specifically with respect to ER stress and its association with different cell death pathways. In our study, we observed that both compounds increase reactive oxygen species (ROS) generation, down regulate mitochondrial membrane potential (MMP) and affect DNA integrity. Our results indicate that Parathion causes ER stress, up regulates the expression of GRP78, leads to nuclear localization of p53 and induces autophagy while Rotenone down regulates GRP78, causes cytoplasmic localization of p53 and inhibits autophagy. Therefore, it may be concluded that GRP78 affects p53 localization which in turn regulates autophagy.

Novel protein-protein interactions of TPPII, p53, and SIRT7.

Novel protein-protein interactions of TPPII, SIRT7, and p53 were detected by co-immunoprecipitation using both HeLa cell lysates and the cytoplasmic fraction prepared by fractionation of mouse liver tissue. The interactions were further verified in vivo by in situ proximity ligation assay (PLA) within control HEK293 cells transformed with empty vector, highactTPPII HEK293 cells over-expressing murine TPPII displaying high specific enzymatic activity and in lowactTPPII HEK293 cells over-expressing human TPPII having low specific activity of the enzyme. Besides an abundant cytoplasmic localization of TPPII-p53 interaction signal, the nuclear interactions were also demonstrated. The cytoplasmic interactions were likewise detected between TPPII and SIRT7 in control HEK293 and lowactTPPII HEK293 cells. The interactions of SIRT7 with p53 were confirmed in three HEK293 cell transformants as well. The cytoplasmic occurrence of SIRT7 protein was demonstrated by immunofluorescence, when both nucleolar and cytoplasmic signals were identified within HEK293 cells and primary human fibroblasts. The unique cytoplasmic localization of SIRT7 protein was discussed based on an epitope specificity of N-terminus specific SIRT7 antibodies utilized in the present study compared with C-terminus specific antibodies previously used for nuclear detection of SIRT7 by other authors. The epitope sequence of N-terminal antibodies is occurring in all three splicing variants of SIRT7 compared to the epitope of C-terminal antibody, which is specific exclusively to the splicing variant 1. The cytoplasmic localization of p53 detected by immunofluorescence supported the results from its interactions with TPPII and SIRT7 observed by in situ PLA within model cells. Novel interactions of TPPII, p53, and SIRT7 presented in this study might contribute to the knowledge of the regulatory effects of these proteins on apoptotic pathways and to the understanding mechanisms of aging and lifespan regulation.

Host response during Yersinia pestis infection of human bronchial epithelial cells involves negative regulation of autophagy and suggests a modulation of survival-related and cellular growth pathways.

Yersinia pestis (Yp) causes the re-emerging disease plague, and is classified by the CDC and NIAID as a highest priority (Category A) pathogen. Currently, there is no approved human vaccine available and advances in early diagnostics and effective therapeutics are urgently needed. A deep understanding of the mechanisms of host response to Yp infection can significantly advance these three areas. We employed the Reverse Phase Protein Microarray (RPMA) technology to reveal the dynamic states of either protein level changes or phosphorylation changes associated with kinase-driven signaling pathways during host cell response to Yp infection. RPMA allowed quantitative profiling of changes in the intracellular communication network of human lung epithelial cells at different times post infection and in response to different treatment conditions, which included infection with the virulent Yp strain CO92, infection with a derivative avirulent strain CO92 (Pgm-, Pst-), treatment with heat inactivated CO92, and treatment with LPS. Responses to a total of 111 validated antibodies were profiled, leading to discovery of 12 novel protein hits. The RPMA analysis also identified several protein hits previously reported in the context of Yp infection. Furthermore, the results validated several proteins previously reported in the context of infection with other Yersinia species or implicated for potential relevance through recombinant protein and cell transfection studies. The RPMA results point to strong modulation of survival/apoptosis and cell growth pathways during early host response and also suggest a model of negative regulation of the autophagy pathway. We find significant cytoplasmic localization of p53 and reduced LC3-I to LC3-II conversion in response to Yp infection, consistent with negative regulation of autophagy. These studies allow for a deeper understanding of the pathogenesis mechanisms and the discovery of innovative approaches for prevention, early diagnosis, and treatment of plague.

Diabetes-induced oxidative DNA damage alters p53-p21CIP1/Waf1 signaling in the rat testis.

Diabetes is increasingly becoming a major cause of large-scale morbidity and mortality. Diabetes-induced oxidative stress alters numerous intracellular signaling pathways. Although testicular dysfunction is a major concern in diabetic men, the mechanistic alterations in the testes that lead to hypogonadism are not yet clear. Oxidative mitochondrial DNA damage, as indicated by 7,8-dihydro-8-oxo-2'-deoxyguanosine, and phosphorylation of p53 at ser315 residue (p-p53ser315) increased in a stage- and cell-specific manner in the testes of rats that were diabetic for 1 month (DM1). Prolongation of diabetes for 3 months (DM3) led to an increase in nuclear oxidative DNA damage in conjunction with a decrease in the expression of p-p53ser315. The nuclei of pachytene and preleptotene spermatocytes, steps 1, 11, and 12 spermatids, secondary spermatocytes and the Sertoli cells, and the meiotic figures showed an increase in the expression of p-p53ser315. An increase in the expression of a downstream target of p53 and protein 21(cyclin-dependent kinase interacting protein 1/wild-type p53-activated factor 1) (p21(CIP1/Waf1)) in both diabetic groups did not show any time-dependent effects but occurred concurrent with an upregulation of p-p53ser315 in DM1 and a downregulation of the protein in DM3. In diabetic groups, the expression of p21(CIP1/Waf1) was mainly cytoplasmic but also perinuclear in pachytene spermatocytes and round spermatids. The cytoplasmic localization of p21(CIP1/Waf1) may be suggestive of an antiapoptotic role for the protein. The perinuclear localization is probably related to the cell cycle arrest meant for DNA damage repair. Diabetes upregulates p21(CIP1/Waf1) signaling in testicular germ cells in association with alteration in p-p53ser315 expression, probably to counteract DNA damage-induced cell death.

The cytoplasmic side of p53's oncosuppressive activities.

The tumor suppressor p53 is a transcription factor that in response to a plethora of stress stimuli activates a complex and context-dependent cellular response ultimately protecting genome integrity. In the last two decades, the discovery of cytoplasmic p53 localization has driven an intense research on its extra-nuclear functions. The ability to induce apoptosis acting directly at mitochondria and the related mechanisms of p53 localization and translocation in the cytoplasm and mitochondria have been dissected. However, recent works indicate the involvement of cytoplasmic p53 also in biological processes such as autophagy, metabolism, oxidative stress and drug response. This review will focus on the mechanisms of cytoplasmic p53 activation and the pathophysiological role of p53's transcription-independent functions, highlighting possible therapeutic implications.

Nrf2 Deficiency Leads to Reduced Hematopoietic Stem Cell Self-Renewal and Increased Sensitivity to Genotoxic Stressors Through Impaired P53 Function

Abstract 397Nrf2 is a basic region-leucine-zipper transcription factor that regulates expression of numerous genes required for oxidative stress response, drug and toxin detoxification, protein ubiquitination, and proteasomal degradation of damaged proteins. Therefore Nrf2 represents a major cellular defense mechanism against oxidative and toxic stresses. We have shown that CD34+ hematopoietic stem/progenitor cells (HSPC) drawn from peripheral blood stem cell (PBSC) autografts of lymphoma patients who develop therapy-related myelodysplasia or acute myeloid leukemia (t-MDS/AML) after autologous hematopoietic cell transplantation (aHCT) demonstrate significantly reduced expression of Nrf2-mediated stress response pathway genes compared to cells from patients who do not develop t-MDS/AML (Cancer Cell 20, 591–605). We have extended these findings to study the role of Nrf2 in HSPC regulation and the response to genotoxic chemotherapeutic agents by using an Nrf2 knockout (KO) mouse model. Nrf2 KO mice demonstrated impaired bone marrow (BM) long-term hematopoietic stem cell (LT-HSC) function, manifested by significantly reduced competitive engraftment of BM cells in recipient mice at 16 weeks compared to wild-type (WT) controls (42.5±2.8% vs. 57.2±3.3% peripheral blood engraftment for KO and WT donors respectively, p<0.01). An even more significant defect in engraftment of KO cells was seen after transplantation to secondary recipients (16.4±1.7% vs. 61.9±1.5% peripheral blood engraftment at 8 weeks for KO and WT donors respectively, p<0.01). Nrf2 KO mice also demonstrated increased sensitivity to ENU treatment compared to WT mice. ENU (100mg/kg) treatment resulted in severe anemia at 7 months in 100% of KO mice compared with 36% of WT mice (p<0.001). Furthermore, anemia developed at a median of 5 months in Nrf2 KO mice compared to 10 months in WT controls. Pathological examination of BM from anemic mice revealed reduced erythropoiesis with impaired erythroid maturation and expansion of myeloid cells, consistent with myeloid dysplasia. Q-RT-PCR analysis demonstrated significantly reduced expression of Nrf2 target genes, including Hmo-1, Nqo-1, Gclc and Gpx1, in BM c-kit+ HSPC from KO mice compared to WT mice (p<0.05). Interestingly, expression of several P53 target genes including Bax, Puma, Cdkn1a and Necdin were also significantly reduced in BM c-kit+ cells from Nrf2 KO compared to WT mice. We also observed reduced P53 target gene expression in Nrf2 KO c-Kit cells compared with WT cells 4 hours after treatment with ENU (p<0.05), indicating impaired P53 response in Nrf2 deficient HSPC. To examine the P53 response to DNA damaging agents in LT-HSC, we exposed Nrf2 KO and WT mice to 2Gy irradiation and selected BM LT-HSC (Lin-Sca-1+c-kit+Flt-3-CD150+CD48-) using flow cytometry 12 hours after irradiation. Q-RT-PCR analysis showed significantly reduced expression of P53 target genes Bax, Puma and Necdin (p<0.05), and a trend towards reduced expression of Cdkn1a (p=0.08), in Nrf2 KO compared to WT LT-HSC. Immunofluorescence microscopy showed significantly reduced nuclear and increased cytoplasmic localization of P53 in Nrf2 KO compared to WT LT-HSC. Western blotting analysis showed increased levels of ubquitinated proteins in BM c-kit+ HSPC from Nrf2 KO compared to WT mice, and immunoprecipitation followed by Western blotting for P53 revealed increased high molecular weight bands indicative of ubiquitinated P53 in Nrf2 KO HSPC. Nrf2 is a key regulator of protein ubiquitination and proteasomal degradation, and these results suggest that abnormal cytoplasmic accumulation of ubiquitinated P53 may contribute to altered P53 function in Nrf2 deficient HSPC. In conclusion, our results show that Nrf2 deficiency results in impaired HSC self-renewal capacity under physiological conditions, and significantly increased sensitivity to genotoxic stress, potentially explained by altered P53 protein modulation in Nrf2 deficient HSPC. These observations suggest that Nrf2 deficiency could contribute to development of t-MDS/AML following exposure to genotoxic agents, and support further evaluation of Nrf2 as a potential target of chemopreventive efforts. Disclosures:No relevant conflicts of interest to declare.

Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen Interacts with Multifunctional Angiogenin To Utilize Its Antiapoptotic Functions

Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with the angioproliferative Kaposi's sarcoma (KS). KSHV infection and the expression of latency-associated nuclear antigen (LANA-1) upregulates the angiogenic multifunctional 123-amino-acid, 14-kDa protein angiogenin (ANG), which is detected in KS lesions and in KSHV-associated primary effusion lymphoma (PEL) cells. ANG knockdown or the inhibition of ANG's nuclear translocation resulted in decreased LANA-1 gene expression and reduced KSHV-infected endothelial and PEL cell survival (Sadagopan et al., J. Virol. 83:3342-3364, 2009). Further studies here demonstrate that LANA-1 and ANG colocalize and coimmunoprecipitate in de novo infected endothelial cells and in latently infected PEL (BCBL-1 and BC-3) cells. LANA-1 and ANG interaction occurred in the absence of the KSHV genome and other viral proteins. In gel filtration chromatography analyses of BC-3 cell lysates, ANG coeluted with LANA-1, p53, and Mdm2 in high-molecular-weight fractions, and LANA-1, p53, and Mdm2 also coimmunoprecipitated with ANG. LANA-1, ANG, and p53 colocalized in KSHV-infected cells, and colocalization between ANG and p53 was also observed in LANA-1-negative cells. The deletion constructs of ANG suggested that the C-terminal region of amino acids 104 to 123 is involved in LANA-1 and p53 interactions. Silencing ANG or inhibiting its nuclear translocation resulted in decreased nuclear LANA-1 and ANG levels, decreased interactions between ANG-LANA-1, ANG-p53, and LANA-1-p53, the induction of p53, p21, and Bax proteins, the increased cytoplasmic localization of p53, the downregulation of Bcl-2, the increased cleavage of caspase-3, and the apoptosis of cells. No such effects were observed in KSHV-negative BJAB cells. The phosphorylation of p53 at serine 15, which is essential for p53 stabilization and for p53's apoptotic and cell cycle regulation functions, was increased in BCBL-1 cells transduced with short hairpin RNA targeting ANG. Together, these studies suggest that the antiapoptosis observed in KSHV-infected cells and the suppression of p53 functions are mediated in part by ANG, and KSHV has probably evolved to utilize angiogenin's multiple functions for the maintenance of its latency and cell survival. Thus, targeting ANG to induce the apoptosis of cells latently infected with KSHV is an attractive therapeutic strategy against KSHV infection and associated malignancies.

Insulin Receptor Tyrosine Kinase Substrate Enhances Low Levels of MDM2-Mediated p53 Ubiquitination

The tumor suppressor p53 controls multiple cellular functions including DNA repair, cell cycle arrest and apoptosis. MDM2-mediated p53 ubiquitination affects both degradation and cytoplasmic localization of p53. Several cofactors are known to modulate MDM2-mediated p53 ubiquitination and proteasomal degradation. Here we show that IRTKS, a novel IRSp53-like protein inhibited p53-induced apoptosis and depressed its transcription activity. IRTKS bound directly to p53 and increased p53 ubiquitination and cytoplasmic localization. Further studies revealed that IRTKS interacted with MDM2 and promoted low levels of MDM2-mediated p53 ubiquitination in vitro and in vivo. In unstressed cells with low levels of MDM2, IRTKS was found to stabilize the interaction of p53 and MDM2. In stressed cells, IRTKS dissociated from p53, and high levels of MDM2 induced by p53 activation mediate IRTKS poly-ubiquitination and subsequent proteasomal degradation. These data suggest that IRTKS is a novel regulator of p53, modulating low level of MDM2-mediated p53 ubiquitination in unstressed cells.

Cytoplasmic p27 is oncogenic and cooperates with Ras both in vivo and in vitro

p27(Kip1) (p27) can have opposing roles during malignant transformation depending on cellular context: on one hand it functions as a tumor suppressor by inhibiting cyclin-cyclin-dependent kinase (CDK) activity in the nucleus and on the other it may adopt an oncogenic role that is less well understood. To gain further insight into the roles played by p27 during tumorigenesis, we compared the susceptibility with urethane-induced tumorigenesis of two p27 mouse models, p27(-/-) and p27(CK-) knockin, in which p27 cannot bind or inhibit cyclin-CDKs. In this K-Ras-driven tumorigenesis model, p27(CK-) mice had an increase in both tumor number and aggressiveness compared with p27(-/-), indicating a cooperation between p27(CK-) and activated Ras. In the lung, increased tumorigenesis was associated with cytoplasmic localization of p27(CK-) and bronchiolaveolar stem cell amplification. The ability of p27(CK-) to cooperate with other oncogenes was not universal. When c-Myc was used as a transforming agent, p27 status became irrelevant and c-Myc was equally potent in transforming p27(+/+), p27(-/-) and p27(CK-) cells. In fact, c-Myc induced the degradation of wild-type p27 via the Skp-Cullin-F-box (SCF)-Skp2 pathway. In contrast, p27(CK-) levels were not affected by c-Myc expression, as p27(CK-) is insensitive to Skp2-mediated degradation because of its inability to bind cyclin E/CDK2. However, in presence of c-Myc, p27(CK-) remained mostly nuclear, providing an explanation for its inability to cooperate with Myc during transformation. Thus, we propose that the p27(CK-) protein needs to be localized in the cytoplasm in order to function as an oncogene, otherwise it just behaves similar to a null allele.

Silencing Kinase-Interacting Stathmin Gene Enhances Erlotinib Sensitivity by Inhibiting Ser10 p27 Phosphorylation in Epidermal Growth Factor Receptor–Expressing Breast Cancer

The epidermal growth factor receptor (EGFR) signaling pathway has emerged as a promising target for cancer therapy. EGFR tyrosine kinase inhibitors (TKI) such as erlotinib have been approved for cancer treatment but have shown very limited activity in breast cancer patients. Clarifying the molecular mechanism underlying resistance to EGFR TKIs could lead to more effective treatment against breast cancer. We previously reported that the sensitivity of breast cancer cells to erlotinib is partially dependent on p27 and that cytoplasmic localization of p27 is associated with erlotinib resistance. In the present study, we found that erlotinib induces p27 phosphorylation at Ser¹⁰ (S10), and S10 p27 phosphorylation leads to erlotinib resistance in EGFR-expressing breast cancer. Inhibiting S10 phosphorylation of p27 by knocking down human kinase-interacting stathmin (KIS), a nuclear protein that can phosphorylate p27 at S10, led to p27 accumulation in the nucleus and enhanced erlotinib-mediated cytotoxicity. Further, in vivo KIS gene silencing enhanced the antitumor activity of erlotinib in an orthotopic breast cancer xenograft model. KIS depletion also enhanced erlotinib sensitivity in erlotinib-resistant EGFR-expressing triple-negative breast cancer cells. Our study provides a rationale for the development of combinations of erlotinib with KIS inhibition to overcome EGFR TKI resistance in EGFR-expressing breast cancer.

Immunohistochemical evaluation of inflammatory and proliferative markers in adjacent normal thyroid tissue in patients undergoing total thyroidectomy: results of a preliminary study

BackgroundTotal thyroidectomy is the treatment of choice in the majority of thyroid malignancies, preventing the risk of reoperative surgery due to recurrences. In order to assess the usefulness of such an approach, expression levels of inflammatory and proliferative markers were evaluated immunohistochemically in non-lesional adjacent thyroid tissues from a group of patients who underwent total thyroidectomy for different thyroid diseases.MethodsNineteen consecutive patients treated by total thyroidectomy for different thyroid diseases entered the study. IL-6Rb gp130 component of the IL-6 cytokine family members receptor complexes, STAT3 cytokine signalling transduction and transcription activation factor, p53 as tumour suppressor and CK19 cytokeratin as proliferation marker were analyzed in non-lesional thyroid tissues.ResultsGp 130 expression was detected in all tissue samples with a scattered distribution while STAT3 and p53 positivity was observed in 17 out of 19 patients with a prevailing cytoplasmic localization. Cytokeratin 19 positivity was found in patients with papillary carcinoma, in one case of follicular adenoma, 3 multinodular goiters and one Basedow disease.ConclusionBased on the results of this preliminary study, it may be concluded that the presence of a persisting cytokine-mediated activation associated with cytoplasmic localization of p53 is frequently observed in different thyroid diseases. Such a process seems to occur in the thyroid gland as a whole. Moreover, STAT3 activation as well as mutant p53 are risk factors for the development of neoplastic diseases. Total thyroidectomy may be supported as an adequate therapeutic approach for all the patients in whom overexpression of cytokine-dependent markers is detected.

653 H. pylori Promotes the Cytoplasmic Localization of P27 in Gastric Cells by cag-Dependent P27 Phosphorylation

653 H. pylori Promotes the Cytoplasmic Localization of P27 in Gastric Cells by cag-Dependent P27 Phosphorylation

Abstract 1972: Silencing kinase interacting stathmin gene enhances erlotinib sensitivity by inhibiting serine 10 p27 phosphorylation in EGFR-expressing breast cancer

Abstract The epidermal growth factor receptor (EGFR) signaling pathway has emerged as a promising target for cancer therapy. EGFR tyrosine kinase inhibitors (TKIs), such as erlotinib and gefitinib, have been approved for lung cancer treatment but have induced limited clinical responses in breast cancer patients even though EGFR is frequently overexpressed in breast cancer. The purpose of this study was to evaluate whether erlotinib sensitivity in EGFR-expressing breast cancer is affected by phosphorylation of p27Kip1 (p27) and depletion of kinase interacting stathmin (KIS), a nuclear protein that phosphorylates p27. We previously reported that the cytoplasmic localization of p27 is associated with erlotinib resistance. In the present study, we tested the expression levels of phosphorylated p27 after erlotinib treatment in EGFR-expressing breast cancer cell lines such as BT-474 and SK-BR-3. Expression of p27 phosphorylated at serine (S) 10 was significantly increased in response to erlotinib. We then transfected phosphoinhibitory S10A-p27 mutant or phosphomimetic S10D-p27 mutant into those cells and found that S10A-p27-transfected cells were more sensitive to erlotinib and S10D-p27-transfected cells were more resistant to erlotinib. KIS binds the C-terminal domain of p27 and phosphorylates it at S10 in vitro and in vivo, resulting in export of p27 from the nucleus to the cytoplasm. To study the role of KIS in erlotinib sensitivity, we tested the basal expression level of KIS in a panel of breast cancer cell lines and found that KIS expression level is low in erlotinib-sensitive cells and high in erlotinib-resistant cells, suggesting that KIS expression level is associated with erlotinib sensitivity. To test whether inhibition of S10-phosphorylated p27 enhances erlotinib's antiproliferative effect, we performed KIS siRNA knockdown in BT-474 and SK-BR-3 cells. KIS siRNA knockdown led to p27 nucleus accumulation and enhanced erlotinib-induced G1 arrest. Furthermore, KIS depletion enhanced the inhibitory effects of erlotinib on proliferation and anchorage-independent growth in breast cancer cells. Most importantly, in an orthotopic breast cancer xenograft model, KIS gene silencing by KIS siRNA intravenous injection led to p27 nuclear accumulation and enhanced the antitumor activity of erlotinib. Further, KIS depletion enhanced erlotinib sensitivity in erlotinib-resistant EGFR-expressing triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468. These results demonstrate that erlotinib-mediated cytotoxicity is regulated by phosphorylation of p27 at S10 and that inhibiting S10 p27 phosphorylation by KIS gene silencing enhances erlotinib sensitivity. Our study provides a rationale for developing combinations of erlotinib with KIS inhibition to overcome EGFR TKI resistance in EGFR-expressing breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1972.

The location of endogenous wild-type p53 protein in 293T and HEK293 cells expressing low-risk HPV-6E6 fusion protein with GFP

The mechanism underlining human papillomaviruses (HPVs) causing cancer has been studied extensively, and it was concluded that the high-risk HPVs' E6 targeted and degraded tumor suppressor protein p53, leading to infected cells malignant transformation. In contrast, the low-risk HPVs only cause proliferative but non-invasive lesions of infected epithelia. Therefore, we hypothesized that low-risk HPVs' E6 might interact with p53 in a different pattern. We used a mammalian green fluorescent protein (GFP) expression system to express HPV-18E6 and HPV-6E6 fusion proteins in wild-type (wt) p53 cell lines, 293T and HEK293 cells, to investigate the traffic and location of E6s and p53. The results indicated GFP-18E6 was mainly expressed in nucleus, whereas GFP-6E6 was expressed exclusively in cytoplasm. Endogenous wt p53 was shown to be localized in the nuclei of cells transfected with GFP- 18E6. Interestingly, for the first time, we observed that p53 was trapped in the cytoplasm and never translocated into the cell nuclei transfected with GFP-6E6. In conclusion, HPV-6E6 was responsible for the cytoplasmic localization of p53. Therefore, our experiments provide a new insight into the pathogenesis of HPV.