Cytoplasmic P53 Research Articles

Invasive lobular carcinoma with extracellular mucin production-a novel pattern of lobular carcinomas of the breast. Clinico-pathological description of eight cases.

Invasive lobular carcinoma of the breast is known to produce intracellular mucin and has been recognized in single-case reports to show extracellular mucin production, as well. This latter morphology is not only rare but must also be under- or misdiagnosed. The aim was to better characterize this entity. Cases of lobular cancers demonstrating extracellular mucin formation were identified in a multi-institutional effort and their clinical and morphologic features were assessed. Immunohistochemistry was used to characterize the E-cadherin-membrane complex, neuroendocrine differentiation, and to some extent, mucin formation. All but one of the eight cases occurred in postmenopausal patients. Extracellular mucin production was present in 5 to 50% of the tumour samples and rarely also appeared in nodal and distant metastases. The tumours were completely E-cadherin negative and showed cytoplasmic p120 positivity. The majority (n=6/8) was also completely negative for β-catenin, but two tumours displayed focal β-catenin positivity in the mucinous area. MUC1 and MUC2 expression was observed in all and 7/8 tumours, respectively; neuroendocrine differentiation was present in only one. Invasive lobular carcinoma with extracellular mucin formation is a rare morphologic variant of lobular carcinoma prone to be misdiagnosed and warranting further studies.

SR 59230A on the expression of MicroRNAs in myocardium of heart failure rats

To investigate the effects of β3-adrenoceptors(β3-AR) inhibitor SR 59230A on MicroRNAs expression in rat myocardium with chronic heart failure and the related mechanisms. One hundred male SD rats were randomly divided into sham operated group(40)and chronic heart failure(CHF)group(60). Coronary artery ligation was used to induce CHF. Then the rats in CHF group were further randomly divided into CHF control group and CHF+SR 59230A group (CHF+SR). Rats in the sham group were divided into sham control group and sham+SR 59230A group (Sham+SR). The rats in Sham+SR group and CHF+SR group were treated with 1 ml SR 59230A(85 mmoL/L in 0.9% saline)twice a day for seven weeks by intraperitoneal injection, while the rats in control groups were injected with the same amount of saline for seven weeks separately. miScript miRNA PCR Arrays were used to determine the expression profile of MicroRNAs. Immunohistochemistry was used to evaluate the distribution of the related proteins in the heart tissue sections. Western blot was used to detect the expressions of nuclear factor-kappaB(NF-κB),p53 and p53-Phospho-Serine 15 in the heart. ①After in vivo blockade of β3-AR by SR 59230A, there were 18MicroRNAs down-regulated in sham control group and CHF control group. Within them, 6 MicroRNAs were related to NF-κB signaling pathway, they were miR-125b-5p,miR-143-3p,miR-145-5p,miR-26a-5p,miR-30a-5p and miR-320-5p. ②Slides from the heart tissue showed that NF-κB was distributed both in nucleus and cytoplasm, while p53 in cytoplasm was more than that in nucleus in heart tissue sections. The expressions of NF-κB and p53 were higher in the CHF control group than those in the sham control group(P<0.05), but were lower in CHF+SR group than those in CHF control group(P<0.05),while they were elevated in Sham+SR group compared to the sham control group(P<0.05). ③ Compared with the sham control group, the protein expression of NF-κB p65 was increased significantly in the CHF control group (P<0.05). After treated with SR59230A in vivo,the protein expressions of NF-κB and p53-Phospho-Serine 15 were decreased significantly in CHF rats(P<0.05),while the protein expressions of NF-κB, p53 and p53-Phospho-Serine 15 proteins were increased in the sham rats (P<0.05). Blocking of β3-AR improved the damaged heart in CHF rats; β3-AR caused the change of MicroRNAs expression, and it related to NF-κB signal pathway.

P27Kip1 promotes invadopodia turnover and invasion through the regulation of the PAK1/Cortactin pathway

p27Kip1 (p27) is a cyclin-CDK inhibitor and negative regulator of cell proliferation. p27 also controls other cellular processes including migration and cytoplasmic p27 can act as an oncogene. Furthermore, cytoplasmic p27 promotes invasion and metastasis, in part by promoting epithelial to mesenchymal transition. Herein, we find that p27 promotes cell invasion by binding to and regulating the activity of Cortactin, a critical regulator of invadopodia formation. p27 localizes to invadopodia and limits their number and activity. p27 promotes the interaction of Cortactin with PAK1. In turn, PAK1 promotes invadopodia turnover by phosphorylating Cortactin, and expression of Cortactin mutants for PAK-targeted sites abolishes p27's effect on invadopodia dynamics. Thus, in absence of p27, cells exhibit increased invadopodia stability due to impaired PAK1-Cortactin interaction, but their invasive capacity is reduced compared to wild-type cells. Overall, we find that p27 directly promotes cell invasion by facilitating invadopodia turnover via the Rac1/PAK1/Cortactin pathway.

Cellular interactions of the phosphorylated form of AKT in prostate cancer

Phospho-Akt (P-Akt1) promotes proliferation and increased survival in vitro and plays an important role in prostate cancer (PCa) progression as well as the prediction of the probability of recurrence. In this study, the goal was to demonstrate the involvement and impact of P-Akt1 on cellular interactions, biomechanisms, and pathways in PCa. Tissue microarrays from 640 PCa patients were immunostained with various antibodies. Ki-67 was used to measure proliferation index, and terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling was used for apoptotic index. Increased expression of P-Akt1 was associated with an increased proliferation but inversely correlated with apoptotic index. Higher levels of P-Akt1 are associated with both higher levels of cytoplasmic p27 and higher levels of nuclear p27, suggesting an involvement in both cytoplasmic entrapment and phosphorylation of p27. P-Akt1 expression significantly correlated with nuclear and cytoplasmic staining of FHKR and GSK. The strongest correlations were found with the P- forms of both, suggesting enzyme kinetics in the latter. Here, phosphorylation is the principal method of FHKR and GSK inactivation. P-Akt1 correlated with nuclear transcription factor kappa B, suggesting a role in the inhibition through phosphorylation of nuclear transcription factor kappa B. The results of the current study are unique because of the scope of the markers and the size of the population used. In vitro- and in vivo-derived information of P-Akt1 and its downstream effectors demonstrates significant involvement in PCa. Our data suggest that PCa uses multiple mechanisms to regulate this pathway and substantiate the concept of redundancy in cancer pathway regulation. Consequently, new hypothesis-driven studies can be derived from this information.

Removal of ribonucleotides by p53 protein incorporated during DNA synthesis by HIV-1 reverse transcriptase.

HIV-1 reverse transcriptase frequently incorporates ribonucleotides into the proviral DNA in macrophages, but not in lymphocytes. The enzyme exerts an efficient ribonucleotide-terminated primer extension capacity. Furthermore, ribonucleotide-editing repair is attenuated in macrophages. Tumor suppressor p53 protein, displaying an intrinsic 3'→5' exonuclease activity, was found to be involved in efficient proofreading of base-base mismatches produced during DNA synthesis. As the presence of proofreading activity is cardinal for the DNA synthesis accuracy, it was of interest to assess whether p53 can serve as a trans-acting proofreader for HIV-1 reverse transcriptase during ribonucleotide incorporation. We investigated the potential involvement of cytoplasmic p53 in error correction during insertion of ribonucleotides into DNA by recombinant HIV-1 reverse transcriptase in a p53-proficient and deficient background. Primer extension reactions were carried out to elucidate the incorporation and removal of ribonucleotides. The biochemical studies suggest that p53 is involved in a ribonucleotide damage-associated repair mechanism through its capacity to remove preformed 3'-terminal ribonucleotides, to decrease ribonucleotide incorporation and to prevent the 3'-ribo-terminated primer extension during ongoing DNA synthesis by HIV-1 reverse transcriptase. A positive correlation exists between the presence of endogenous p53 and decrease in stable incorporation of ribonucleotides into DNA with p53-harboring lysates of HCT116 cells. p53, by preferential removal of purine over pyrimidine ribonucleotides, may affect the ribonucleotide mutation spectra produced by HIV-1 reverse transcriptase. The data implies that p53 can excise incorrect sugar in addition to base mispairs, thereby expanding the role of p53 in the repair of nucleic acids replication errors.

Rare Case of Painless Jaundice

Cervical cancer remains a major cause of morbidity and mortality. The vast majority are related to human papillomavirus infection which is ubiquitous in the population and results in approximately 12,710 new cases and 4,290 deaths in the US annually, making it the third most common gynecological cancer diagnosis and cause of death. Squamous cell carcinoma and adenocarcinoma account for approximately 69% and 25% of cases of cervical carcinoma, respectively. Cervical cancer may spread to adjacent organs via direct extension or to distant sites through lymphatic or hematogenous dissemination. The most common sites of hematogenous spread are the lung, liver, and bone. Here we present a case of cervical cancer with metastasis to the biliary system without liver metastasis causing common bile duct obstruction with jaundice. A 63-year-old woman with history of cervical cancer presented with painless jaundice. She was diagnosed four months prior with stage IV-A cervical carcinoma and underwent chemotherapy with cisplatin and whole pelvic radiation. After completing five courses of brachytherapy, the last of which was one month prior to admission, she was determined to be in clinical remission. Upon admission, MRCP was obtained and demonstrated evidence of biliary obstruction. ERCP then showed high-grade stenosis of the proximal CBD and beading of the intrahepatic bile ducts, which was treated with stenting. ERCP brushings confirmed metastatic disease with atypical epithelial cells consistent with metastatic SCC. Immunohistochemical studies demonstrated strong and diffuse cytoplasmic and nuclear p16 staining while MIB-1 staining was present in 40-50% of the neoplastic cells. Metastatic cervical carcinoma is a devastating disease with a grim prognosis. Although the cancer typically spreads by direct extension, distant metastases to the lung, liver, and bone have been described. Per our literature review, there has been a single case of metastasis to the ampulla of Vater but none to the bile ducts itself. This patient with a history of stage IV-A cervical carcinoma presented with painless jaundice and was subsequently diagnosed with bile duct metastasis. Unfortunately, the patient was not a candidate for surgical or chemotherapy treatment and was sent to hospice due to her functional status. Fortunately, the incidence and mortality rate of cervical cancer has declined in recent years in the US due to increased cervical cancer screening vigilance.Figure 1Figure 2Figure 3

P53 in the mitochondria, as a trans-acting protein, provides error-correction activities during the incorporation of non-canonical dUTP into DNA.

Mutations in mitochondrial DNA is an outcome of errors produced by DNA polymerase γ during replication and failure of the repair mechanism. Misincorporation of non-canonical dUTP leads to mutagenesis or apoptosis, and may contribute to the cytotoxic effects of 5′-fluorouracil chemotherapy. Tumor suppressor p53 protein in the mitochondria displays physical and functional interactions with mitochondrial DNA and polymerase γ, and by its intrinsic 3′→5′ exonuclease activity can diminish the polymerization errors. Here we demonstrate the impact of p53 on incorporation of uracil into DNA examined with mitochondrial fractions, as the source of polymerase γ. p53 in mitochondria facilitates DNA damage repair functions resulting from uracil–DNA misincorporation. Our biochemical studies revealed that the procession of U:A and mismatched U:G lesions enhances in the presence of recombinant or endogenous cytoplasmic p53. p53 in mitochondria can function as an exonuclease/proofreader for polymerase γ by either decreasing the incorporation of non-canonical dUTP into DNA or by promoting the excision of incorporated nucleotide from nascent DNA, thus expanding the spectrum of DNA damage sites exploited for proofreading as a trans-acting protein. The data suggest that p53 may contribute to defense of the cells from consequences of dUTP misincorporation in both normal and tumor cells.

Molecular mechanism leading to SAHA-induced autophagy in tumor cells: evidence for a p53-dependent pathway

BackgroundRecent studies indicated that histone deacetylase inhibitors (HDACi), a class of anticancer agents, are in addition to their ability of apoptosis induction also capable of provoking autophagy. Promoted by the treatment of malignant uterine sarcoma cells with the HDACi suberoylanilide hydroxamic acid (SAHA), we previously demonstrated predominant dose-dependent activation of autophagy in ESS-1 cells, but prevalent induction of apoptosis in MES-SA cells.MethodsIn order to extend our previous studies, SAHA-treated ESS-1 and MES-SA cells were monitored for protein expression to reveal differences in known markers of apoptosis explaining the different cytotoxic responses. Further analysis of the identified candidate protein included cell rescue experiments by gene transfer followed by subsequent screening of cells for induction of apoptosis and autophagy by immunoblotting, caspase activity as well as LC3 and MDC/PI staining. LDH release assays were performed to assess the amount of cell-mediated cytotoxicity.ResultsIn our search for responsible autophagic regulatory genes upstream of mammalian target of rapamycin (mTOR), we now discovered that, in contrast to MES-SA cells, a TP53-637C>T nonsense mutation located in the transactivating domain of the oncogenic suppressor p53 causes loss of its protein and consequently reduced PUMA induction in ESS-1 cells. Upon re-introduction of wild-type TP53, SAHA-treated ESS-1 cells underwent immediate apoptotic cell death as supported by upregulation of PUMA and caspase-9 as well as by activation of caspases-3 and -7 and PARP-1 cleavage. Concurrent downregulation of autophagy was noticed by upregulated mTor and phospho-mTOR expression as well as monitoring autophagosome formation employing LC3 and MDC staining. Previously, cytoplasmic master regulatory activities of the oncogenic suppressor p53 in inhibiting autophagy and triggering apoptosis were unravelled. Accordingly, p53-deficiency could explain both, the previously documented apoptosis resistance and prevailing SAHA-induced autophagy in ESS-1 cells. Using MES-SA cells with RNAi-silenced p53 expression and several p53-deficient tumor cell lines undergoing SAHA-induced autophagy, we could generally validate our finding suggesting an inhibitory role for p53 in the autophagic pathway in response to SAHA treatment.ConclusionsConclusively, these results could identify cytoplasmic p53 protein as a molecular switch that directly mediates the cytotoxic response of SAHA and thus open new therapeutic avenues.

Prognostic relevance of autophagy-related markers LC3, p62/sequestosome 1, Beclin-1 and ULK1 in colorectal cancer patients with respect to KRAS mutational status.

BackgroundAutophagy is a cellular pathway that regulates transportation of cytoplasmic macromolecules and organelles to lysosomes for degradation. Autophagy is involved in both tumorigenesis and tumour suppression. Here we investigated the potential prognostic value of the autophagy-related proteins Beclin-1, p62, LC3 and uncoordinated (UNC) 51-like kinase 1 (ULK1) in a cohort of colorectal cancer (CRC) specimens.MethodsIn this study, we analysed the immunoexpression of the autophagy-related proteins p62, LC3, Beclin-1 and ULK1 in 127 CRC patients with known KRAS mutational status and detailed clinical follow-up.ResultsSurvival analysis of p62 staining showed a significant correlation of cytoplasmic (not nuclear) p62 expression with a favourable tumour-specific overall survival (OS). The prognostic power of cytoplasmic p62 was found in the KRAS-mutated subgroup but was lost in the KRAS wildtype subgroup. Survival analysis of Beclin-1 staining did not show an association with OS in the complete cohort. LC3 overexpression demonstrated a slight, though not significant, association with decreased OS. Upon stratifying cases by KRAS mutational status, nuclear (not cytoplasmic) Beclin-1 staining was associated with a significantly decreased OS in the KRAS-mutated subgroup but not in the KRAS wildtype CRCs. In addition, LC3 overexpression was significantly associated with decreased OS in the KRAS-mutated CRC subgroup. ULK1 expression was not correlated to survival.ConclusionsImmunohistochemical analyses of LC3, p62 and Beclin-1 may constitute promising novel prognostic markers in CRC, especially in KRAS-mutated CRCs. This strategy might help in identifying high-risk patients who would benefit from autophagy-related anticancer drugs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12957-016-0946-x) contains supplementary material, which is available to authorized users.

Abstract 2445: Cytoplasmic mislocalization of p27 promotes metastasis and its autoantibody correlates with prognosis in osteosarcoma

Abstract Development of metastasis is the major cause of death in osteosarcoma, which is the most common malignant bone tumor in children and young adults. However, detection of metastasis relies solely on imaging techniques and targeted treatments of metastatic patients are still not available to improve the dismal outcome. Using an immunoproteomic approach, we identified that p27 autoantibody was significantly elevated in the plasma of high-risk osteosarcoma patients. Results of immunohistochemistry showed that cytoplasmic mislocalization of p27 occurred in a majority of osteosarcoma cases and in highly metastatic osteosarcoma cell lines. We demonstrated that ectopic expression of cytoplasmic p27 and shRNA-mediated gene silencing promoted and inhibited, respectively, the migration and invasion of osteosarcoma cells. Additionally, a mutation in S10 or T198, but not T157, phosphosite abolished the pro-migration and invasion phenotypes of cytoplasmic p27, possibly through a RhoA-related mechanism. Furthermore, mice injected with cells carrying cytoplasmic p27 increased the development of pulmonary metastasis when compared to an empty vector control. Lastly, using a large cohort of serum samples (n = 233), we showed that a higher level of the p27 autoantibody significantly correlated with poor overall and event-free survival of osteosarcoma (p &amp;lt; 0.05). Together, our results suggest that the circulating p27 autoantibody can be used as a prognostic biomarker at diagnosis and cytoplasmic mislocalization of p27 is a frequent event in osteosarcoma, which can promote tumor cell motility and invasiveness as well as metastasis formation. Targeting specific p27 phosphorylations may provide a novel therapeutic approach for osteosarcoma patients with metastasis. Citation Format: Manjula Nakka, Yiting Li, Aaron J. Kelly, Ching C. Lau, Mark Krailo, Donald A. Barkauskas, John Hicks, Tsz-Kwong Man. Cytoplasmic mislocalization of p27 promotes metastasis and its autoantibody correlates with prognosis in osteosarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2445.

Salidroside protects cortical neurons against glutamate-induced cytotoxicity by inhibiting autophagy.

Recent evidence suggests that glutamate-induced cytotoxicity contributes to autophagic neuron death and is partially mediated by increased oxidative stress. Salidroside has been demonstrated to have neuroprotective effects inglutamate-induced neuronal damage. The precise mechanism of its regulatory role in neuronal autophagy is, however, poorly understood. This study aimed to probe the effects and mechanisms of salidroside in glutamate-induced autophagy activation in cultured rat cortical neurons. Cell viability assay, Western blotting, coimmunoprecipitation, and small interfering RNA were performed to analyze autophagy activities during glutamate-evoked oxidative injury. We found that salidroside protected neonatal neurons from glutamate-induced apoptotic cell death. Salidroside significantly attenuated the LC3-II/LC3-I ratio and expression of Beclin-1, but increased (SQSTM1)/p62 expression under glutamate exposure. Pretreatment with 3-methyladenine (3-MA), an autophagy inhibitor, decreased LC3-II/LC3-I ratio, attenuated glutamate-induced cell injury, and mimicked some of the protective effects of salidroside against glutamate-induced cell injury. Molecular analysis demonstrated that salidroside inhibited cortical neuron autophagy in response to glutamate exposure through p53 signaling by increasing the accumulation of cytoplasmic p53. Salidroside inhibited the glutamate-induced dissociation of the Bcl-2-Beclin-1 complex with minor affects on the PI3K/Akt/mTOR signaling pathways. These data demonstrate that the inhibition of autophagy could be responsible for the neuroprotective effects of salidroside on glutamate-induced neuronal injury.

P53 Mediates Colistin-Induced Autophagy and Apoptosis in PC-12 Cells.

The mechanism of colistin-induced neurotoxicity is still unknown. Our recent study (L. Zhang, Y. H. Zhao, W. J. Ding, G. Z. Jiang, Z. Y. Lu, L. Li, J. L. Wang, J. Li, and J. C. Li, Antimicrob Agents Chemother 59:2189-2197, 2015, http://dx.doi.org/10.1128/AAC.04092-14; H. Jiang, J. C. Li, T. Zhou, C. H. Wang, H. Zhang, and H. Wang, Int J Mol Med 33:1298-1304, 2014, http://dx.doi.org/10.3892/ijmm.2014.1684) indicates that colistin induces autophagy and apoptosis in rat adrenal medulla PC-12 cells, and there is interplay between both cellular events. As an important cellular stress sensor, phosphoprotein p53 can trigger cell cycle arrest and apoptosis and regulate autophagy. The aim of the present study was to investigate the involvement of the p53 pathway in colistin-induced neurotoxicity in PC-12 cells. Specifically, cells were treated with colistin (125 μg/ml) in the absence and presence of a p53 inhibitor, pifithrin-α (PFT-α; 20 nM), for 12 h and 24 h, and the typical hallmarks of autophagy and apoptosis were examined by fluorescence/immunofluorescence microscopy and electron microscopy, real-time PCR, and Western blotting. The results indicate that colistin had a stimulatory effect on the expression levels of the target genes and proteins involved in autophagy and apoptosis, including LC3-II/I, p53, DRAM (damage-regulated autophagy modulator), PUMA (p53 upregulated modulator of apoptosis), Bax, p-AMPK (activated form of AMP-activated protein kinase), and caspase-3. In contrast, colistin appeared to have an inhibitory effect on the expression of p-mTOR (activated form of mammalian target of rapamycin), which is another target protein in autophagy. Importantly, analysis of the levels of p53 in the cells treated with colistin revealed an increase in nuclear p53 at 12 h and cytoplasmic p53 at 24 h. Pretreatment of colistin-treated cells with PFT-α inhibited autophagy and promoted colistin-induced apoptosis. This is the first study to demonstrate that colistin-induced autophagy and apoptosis are associated with the p53-mediated pathway.

Hydroquinone-induced FOXP3-ADAM17-Lyn-Akt-p21 signaling axis promotes malignant progression of human leukemia U937 cells

Hydroquinone (1,4-benzenediol; HQ), a major marrow metabolite of the leukemogen benzene, has been proven to evoke benzene-related hematological disorders and myelotoxicity in vitro and in vivo. The goal of the present study was to explore the role of FOXP3 in HQ-induced malignant progression of U937 human leukemia cells. U937 cells were treated with 5μM HQ for 24h, and the cells were re-suspended in serum-containing medium without HQ for 2days. The same procedure was repeated three times, and the resulting U937/HQ cells were maintained in cultured medium containing 5μM HQ. Proliferation and colony formation of U937/HQ cells were notably higher than those of U937 cells. Ten-eleven translocation methylcytosine dioxygenase-mediated demethylation of the Treg-specific demethylated region in FOXP3 gene resulted in higher FOXP3 expression in U937/HQ cells than in U937 cells. FOXP3-induced miR-183 expression reduced β-TrCP mRNA stability and suppressed β-TrCP-mediated Sp1 degradation, leading to up-regulation of Sp1 expression in U937/HQ cells. Sp1 up-regulation further increased ADAM17 and Lyn expression, and ADAM17 up-regulation stimulated Lyn activation in U937/HQ cells. Moreover, U937/HQ cells showed higher Lyn-mediated Akt activation and cytoplasmic p21 expression than U937 cells did. Abolishment of Akt activation decreased cytoplasmic p21 expression in U937/HQ cells. Suppression of FOXP3, ADAM17, and Lyn expression, as well as Akt inactivation, repressed proliferation and clonogenicity of U937/HQ cells. Together with the finding that cytoplasmic p21 shows anti-apoptotic and oncogenic activities in cancer cells, the present data suggest a role of FOXP3/ADAM17/Lyn/Akt/p21 signaling axis in HQ-induced hematological disorders.

Prognostic relevance of autophagy markers LC3B and p62 in esophageal adenocarcinomas.

Esophageal adenocarcinomas (EAC) are aggressive tumors with considerable rates of chemoresistance. Autophagy is a lysosome-dependent degradation process, characterized by the formation of vesicles called autophagosomes, and has been implicated in cancer. Protein light chain 3 B (LC3B) and p62 are associated with autophagosomal membranes and degraded. We aimed to assess the impact of basal autophagy on EAC. In EAC cell lines, an increase in LC3B and p62 was observed with increasing concentrations of the autophagy inhibitor chloroquine, which indicates functional basal autophagy. LC3B and p62 immunohistochemistry was performed on primary resected EAC. High LC3B and p62 expression was associated with earlier tumor stages (p < 0.05). High nuclear and cytoplasmic p62 staining were associated with a better prognosis (p = 0.006; p = 0.028). Various combinations of p62 expression with or without LC3B expression identified different prognostic groups. Tumors with low total p62 (p = 0.007) or low LC3B/low p62 expression had the worst outcome (p = 0.007; p = 0.005). A combination score of dot-like/cytoplasmic p62 and nuclear p62 staining was an independent prognostic parameter (p = 0.033; HR = 0.6). This study highlights the potential significance of basal autophagy in EAC biology. Tumors with low LC3B and p62 expression show the most aggressive behavior and may be candidates for autophagy regulating therapeutics.

Loss of p27 expression is associated with MEN1 gene mutations in sporadic parathyroid adenomas.

MEN1 is the main gene responsible for tumorigenesis of syndromic and sporadic primary hyperparathyroidism (PHPT). Germline mutations of the CDKN1B/p27Kip gene have been associated with multiple endocrine tumors in rats and humans. To evaluate the involvement of the CDKN1B gene and its relationship with MEN1 in sporadic PHPT, we carried out sequencing and loss of heterozygosity analyses of the CDKN1B gene in 147 sporadic parathyroid adenomas. p27 immunohistochemistry and genetic screening of the MEN1 gene were performed in 50 cases. Three germline CDKN1B variants (c.-80C>T, c.-29_-26delAGAG, c.397C>A) were identified in 3/147 patients. Reduction of CDKN1B gene transcription rate was demonstrated in vitro for the novel c.-80C>T and the c.-29_-26delAGAG variants. Loss of p27 expression was detected in the tumor carrying the c.-29_-26delAGAG variant. Two tumors carrying the CDKN1B variants also harbored a MEN1 mutation. Fifty-four percent of 50 CDKN1B mutation-negative tumors had a reduction of p27 nuclear staining. Somatic MEN1 mutations, identified in 15/50 samples, significantly segregated in tumors negative for nuclear and cytoplasmic p27 staining. The germline nature of the CDKN1B mutations suggests that they might predispose to PHPT. The lack of somatic CDKN1B mutations in our samples points to a rare involvement in parathyroid adenomas, despite the frequent loss of nuclear p27 expression. MEN1 biallelic inactivation seems to be directly related to down-regulation of p27 expression through the inhibition of CDKN1B gene transcription.

Suppressing activity of tributyrin on hepatocarcinogenesis is associated with inhibiting the p53-CRM1 interaction and changing the cellular compartmentalization of p53 protein.

Hepatocellular carcinoma (HCC), an aggressive and the fastest growing life-threatening cancer worldwide, is often diagnosed at intermediate or advanced stages of the disease, which substantially limits therapeutic approaches for its successful treatment. This indicates that the prevention of hepatocarcinogenesis is probably the most promising approach to reduce both the HCC incidence and cancer-related mortality. In previous studies, we demonstrated a potent chemopreventive effect of tributyrin, a butyric acid prodrug, on experimental hepatocarcinogenesis. The cancer-inhibitory effect of tributyrin was linked to the suppression of sustained cell proliferation and induction of apoptotic cell death driven by an activation of the p53 apoptotic signaling pathway. The goal of the present study was to investigate the underlying molecular mechanisms linked to tributyrin-mediated p53 activation. Using in vivo and in vitro models of liver cancer, we demonstrate that an increase in the level of p53 protein in nuclei, a decrease in the level of cytoplasmic p53, and, consequently, an increase in the ratio of nuclear/cytoplasmic p53 in rat preneoplastic livers and in rat and human HCC cell lines caused by tributyrin or sodium butyrate treatments was associated with a marked increase in the level of nuclear chromosome region maintenance 1 (CRM1) protein. Mechanistically, the increase in the level of nuclear p53 protein was associated with a substantially reduced binding interaction between CRM1 and p53. The results demonstrate that the cancer-inhibitory activity of sodium butyrate and its derivatives on liver carcinogenesis may be attributed to retention of p53 and CRM1 proteins in the nucleus, an event that may trigger activation of p53-mediated apoptotic cell death in neoplastic cells.

Initial evidence for Sec62 as a prognostic marker in advanced head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a malignancy with an increasing incidence. To aid with the selection of the most appropriate therapy, biomarkers have become a specific research focus. Sec62 is involved in endoplasmic reticulum stress tolerance and cell migration, and has been identified as a novel prognostic marker for non-small cell lung cancer. In addition, Sec62 may be a promising candidate in HNSCC. Pretreatment biopsies of 35 patients with locally advanced HNSCC, who were treated with definitive chemoradiation therapy without prior surgery, were examined for the expression of Sec62 protein, as well as the expression of epidermal growth factor receptor (EGFR), p16 and survivin proteins. Immunohistological results were correlated with patient overall survival (OS) and progression-free survival (PFS) times. In the present patient cohort, 12/35 cases (34%) demonstrated strong and 8/35 cases (23%) moderate Sec62 staining intensity. Additionally, in 11/35 cases (31%), weak staining was observed, and only 4/35 cases (11%) were Sec62-negative. Notably, a high Sec62 protein level was associated with a significantly poorer OS and PFS (P=0.020 and P=0.028, respectively). Furthermore, higher nuclear survivin expression showed a weak trend for poorer OS rate (P=0.079), whilst neither cytoplasmic survivin, EGFR nor p16 influenced OS or PFS significantly. The present study indicated that Sec62 is a promising prognostic marker for HNSCC. Increased Sec62 protein expression may indicate a poorer prognosis in advanced HNSCC. As the present study was focused on patients treated by chemoradiation therapy, further studies with larger patient cohorts and alternative treatment approaches are required in order to define the prognostic value of Sec62 in HNSCC.

Prognostic significance of cytoplasmic p27 in oral squamous cell carcinoma.

p27 is a cell cycle-dependent kinase inhibitor whose presence in nucleus is associated with good prognosis. Recent studies propose that when localized to cytoplasm, it functions as an oncogene and confers a poorer prognosis. This study aimed at analysing the subcellular localization of p27 and its prognostic significance in oral squamous cell carcinomas (OSCCs). Immunohistochemistry for p27 was carried out on 60 cases of OSCC (30 cases each of those with lymph node metastasis [LN+ve SCC] and without lymph node metastasis [LN-ve SCC]) and 30 normal mucosa. The relationship between p27 localization and prognosis was analysed statistically. Nuclear immunopositivity was seen in 15%, 23%, 7% and 60%, while cytoplasmic immunopositivity was seen in 80%, 63%, 97% and 43% of all SCC, LN+ve OSCC, LN-ve SCC cases and normal mucosa, respectively. There was a significant inverse correlation between nuclear and cytoplasmic p27 immunopositivity (P = 0.001). Nodal status and tumour stage were the only two parameters that correlated with disease-free survival (DFS) in OSCC cases. However, in LN+ve SCC, a significantly shortened DFS was seen in cases with cytoplasmic p27 expression compared to those without (P = 0.02). Conversely, LN+ve SCC with nuclear p27 had longer DFS on comparison with those without (P = 0.04). To the best of our knowledge, this is the first study to analyse cytoplasmic localization of p27 in OSCC and correlate with prognosis. Cytoplasmic localization is associated with poor prognosis in OSCC with lymph node metastasis allowing the consideration of cytoplasmic p27 in predicting prognosis and targeted therapeutic approaches.

Interferon- β Augments the Effects of Cisplatin on Hcc Cell Lines by Regulating Sub Cellular Localization of P21

Introduction: Interferon (IFN) has been used for patients with advanced hepatocellular carcinoma (HCC) in combination with chemotherapeutic agents. We previously reported that IFN-? induced a shift into the nucleus of Cytoplasmic p21 (Cip1/WAF1), known as a tumor-suppressor gene, and contributed to the prevention of HCC. Aims: To use hepatoma cell lines to compare the growth-suppression activity of IFN-? and ?, in the context of enhancing cisplatin’s effects. Results: IFN-? caused a significant nuclear localization of p21 when compared to IFN-? IFN-? augmented the cytotoxic effects of cisplatin more than IFN- ?. Notably, when IFN-? was used only for pre-treatment, significant apoptosis with subsequent cisplatin treatment was observed. Conclusion: Results suggest that IFN- ? enhances the cytotoxic effects of cisplatin more than IFN-?, partly because of its effect of keeping p21 in the nucleus. Thus, IFN-?-based chemo-therapeutic regimen might provide more effective outcomes of HCC treatment.

Transient delay of radiation-induced apoptosis by phorbol acetate

The mechanisms of interference of a model tumour promoter 12-O-tetra-decanoylphorbol-13-acetate (TPA) with radiation-induced apoptosis in human peripheral lymphocytes have been investigated. The cells were treated with TPA under various conditions and thereafter exposed to a single lethal dose of gamma radiation. Morphological and biochemical changes characteristic of apoptosis were followed up to 72 h of post-irradiation time. Acute exposure to low concentration of TPA resulted in delay in the onset of radiation-induced apoptosis (determined as morphological changes and rate of mitochondrial demise) by 24-48 h as compared to the irradiated, sham TPA-treated cells. The time course of this delay correlated well with confinement of the p53 protein to the cytoplasm and increase in bcl-2 levels at the nuclear periphery of irradiated cells. Our results indicate that confinement of p53 in the cytoplasm is one of the potential mechanisms by which TPA interferes with the process of radiation-induced apoptosis in human lymphocytes.