Inducible P53 Expression Research Articles

Abstract LB-093: Refolding & restoring function to mutant p53: HDAC inhibitor MS275 partially restores activity to R175H mutant p53

Abstract Introduction: Most p53 mutations occur in the DNA binding domain, resulting not only in a loss of WT p53 function but also in the gain of additional onocogenic properties such as increased cellular proliferation and metastasis. Two newly developed pharmaceuticals, NSC 319726 and SCH 529074, refold mutant p53 to a WT conformation, reduce cell proliferation, decrease aggregate formation, and represent a novel therapeutic approach. Another approach to regulating p53 activity is through acetylation. This post-translational modification leads to an activating conformational change in WT p53, but it is unclear how acetylation affects mutant p53. Two histone deacetylase inhibitors (HDACi), MS275 (Entinostat) and FK228 (Romidepsin), are promising chemotherapeutics that increase acetylation of many proteins. In this study, we ask if treatment with these HDACi leads to refolding of mutant p53, reduction of proliferation, and p53 transcription factor activity. Methods: R175H, H193Y, R248Q, and R273H constructs were transfected into a p53-null cell line. Inducible p53 expression in stable subclones was quantitated by immunofluorescence, western blot, and ELISA. Proliferation was measured by incorporation of EdU after treatment with FK228, MS275, NSC 319726, and SCH 529074. Refolding of p53 was determined before and after treatment using the conformational antibodies pAb240 and pAb1620, which bind to unfolded and folded p53, respectively. Functionality of the R175H mutant p53 was assessed by measuring the ability of p53 to bind its response element. Results: We focused on the R175H conformational p53 mutation as had a higher rate of cellular proliferation measured by EdU incorporation compared to the other mutants (p< 0.05). As expected, NSC 319726 treatment reduces R175H proliferation (p< 0.0001) and promotes refolding into the WT p53 conformation (p< 0.05) while no difference was detected with the broad spectrum mutant p53 reactivator SCH 529074. The HDAC inhibitors MS275 and FK228 reduce R175H proliferation (p< 0.0001) and MS275 appears to promote refolding into the WT p53 conformation while FK228 does not. When compared to control, transcription factor activity increased 3.8-fold with NSC 319726 treatment (p < 0.05), 6.0-fold with MS275 treatment (p< 0.0001), and 4.6-fold with FK228 treatment (p<0.01). Results suggest that HDACi may activate mutant p53 and that MS275 may do so by restoring WT conformation. Conclusion: The HDAC inhibitor MS275 reactivates R175H mutant p53 as measured by increased transcription factor activity apparently through refolding, as determined by pAb1620 binding following treatment. These results imply a novel mode of action for HDACi. Clinically this suggests that MS275 may be able to restore WT p53 tumor suppressor activity. Citation Format: Margaux Baatz, Brianna Flores, Jacob Moore, Kathryn J. Leyva, Elizabeth E. Hull. Refolding & restoring function to mutant p53: HDAC inhibitor MS275 partially restores activity to R175H mutant p53 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-093.

Alterations in the p53-SOCS2 axis contribute to tumor growth in colon cancer

Altered expression of suppressor of cytokine signaling (SOCS) is found in various tumors. However, regulation of SOCS2 by upstream molecules has yet to be clearly elucidated, particularly in tumor cells. SCOCS2 expression was examined in tumor cells transfected with an inducible p53 expression system. The impact of SOCS2 on cell proliferation was measured with in vitro assays. Inhibition of tumorigenicity by SOCS2 knockdown was assessed via a mouse model. Expression profiles were compared and genes differentially expressed were identified using four types of p53-null cells (Saos, HLK3, PC3, and H1299) and the same cells stably expressing p53. Twelve kinds of target genes were simultaneously upregulated or downregulated by p53 in three or more sets of p53-null cells. SOCS2 expression was reciprocally inhibited by inducible p53 expression in p53-null cells, even colon cancer cells. SOCS2 promoter activity was inhibited by wild type but not mutant p53. SOCS2 knockdown inhibited tumor growth in vitro and in an animal xenograph model. SOCS2 overexpression was detected in a murine model of azoxymethane/dextran sulfate sodium-induced colitis-associated colon cancer compared to mock-treated controls. SOCS2 expression was heterogeneously upregulated in some human colon cancers. Thus, SOCS2 was upregulated by p53 dysfunction and seemed to be associated with the tumorigenic potential of colon cancer.

Defining the minimal peptide sequence of the ING1b tumour suppressor capable of efficiently inducing apoptosis.

The ING1b protein is a type-II tumour suppressor and stoichiometric member of the Sin3 histone deacetylase (HDAC) protein complex in which it acts to target HDAC activity to regulate chromatin structure. Altering ING1 levels by ectopic expression of ING1b in cancer cells promotes apoptosis, whereas altering levels by knockout in normal murine fibroblasts alters sensitivity to doxorubicin-induced apoptosis. We have identified a minimal region of ING1b capable of inducing levels of apoptosis in targeted cells as effectively as full-length ING1b, using transient overexpression of ING1b fragments followed by the Annexin V assay. We observed high levels of apoptosis in 14 of 14 cancer cell lines tested. Infecting triple-negative tumorigenic MDA-MB-468 breast cancer, U2OS or Saos-2 cells at multiplicities of infection (MOIs) ranging from 10 to 20 rapidly triggered apoptosis in ~80% of infected cells within 48 h. This was not due to the effects of virus, as infection at the same MOI with a control adenovirus expressing GFP was not effective in inducing apoptosis. When used at low MOIs, the ING1b fragment showed a cell-killing efficacy that was higher than native, full-length ING1b. Using a doxycycline-regulated inducible p53 expression system demonstrated that apoptosis induced by the ING1b fragment was p53 independent. Given the growing importance of combination therapies, we evaluated whether there was synergism between the ING1b fragment and HDAC inhibitors. Combination treatments with TSA, LBH 589 and SAHA reduced cancer cell survival by 3.9–4.7-fold as compared with single-drug treatment, and resulted in ~90% reduction in cell survival. Normalized isobologram analysis confirmed strong synergism between the ING1b fragment and drugs tested. These findings provide support for using ING1b-derived therapeutics as adjuvant treatments in combination with existing epigenetic therapies.

Abstract 2940: Identifying the minimal region of the ING1 tumor suppressor capable of efficiently killing cancer cells

Abstract ING1b is a type II tumor suppressor and a stoichiometric member of HDAC-containing protein complexes. ING1b overexpression promotes apoptosis, and decreased levels of ING1b are frequently observed in human tumors and cancer cell lines. Previously, we reported the identification of the regions of ING1b protein required for its pro-apoptotic function. Using deletion mutant analysis and transient expression in HEK293 cells we have determined that ING1b-derived peptides comprised of the NLS/NTS domain and the third alpha helix (A3H) of ING1b including the N-terminal portion of Lamin Interaction Domain (LID) are able to induce apoptosis at levels comparable to those of the full length ING1b, despite their lacking the PHD domain through which they affect the histone epigenetic code. Here, we report that adenoviral delivery of the A3H-NLS/NTS peptide led to a significant decrease in cell survival and induced apoptosis in a majority of tested cancer cell lines. The list of sensitive cancer cell lines was broad and included lines derived from both primary and metastatic tumor sources. As shown using an Annexin V binding assay, decreased survival of cells infected with adenovirus expressing A3H-NLS/NTS peptide was due to a large number of infected cells undergoing apoptosis. In fact, infecting triple negative tumorigenic MDA-MB-468 breast cancer line with 30 MOI of the Ad-A3H-NLS/NTS virus triggered rapid apoptosis in nearly 70% of infected cells. Importantly, these effects were time and dose dependent. Also, using a cell line carrying an inducible p53-expression system, we established that the ability of Ad-A3H-NLS/NTS to induce apoptosis is p53-independent. Next, we evaluated the cell death inducing properties of Ad-A3H-NLS/NTS using 10 breast cancer cell lines that were chosen based on their sensitivity to common HDAC inhibitors. While the overall sensitivity to Ad-A3H-NLS/NTS induced cell death varied among these lines, we observed no obvious correlation between the sensitivity to Ad-A3H-NLS/NTS and sensitivity/resistance to HDAC inhibitors. These findings suggest that Ad-A3H-NLS/NTS may have a broad application, including in those cancer cells lines that are normally resistant to conventional chemotherapy. Furthermore, combined application of Ad-A3H-NLS/NTS with either Trichostatin A (TSA) or Panobinostat (LBH-589) resulted in a strong additive, and possibly synergistic killing effect. Using the virus with either TSA or LBH-589 increased efficacy by nearly 3.5-fold as compared to the treatment with either TSA or LBH-589 along. Currently, the synergy between the Ad-A3H-NLS/NTS and common chemotherapeutics is being closely evaluated. Our long-term goal is to develop ING1b-based therapeutics that can be used as an adjuvant therapy in combination with existing cancer treatments. Citation Format: Oleksandr Boyko, Karl T. Riabowol. Identifying the minimal region of the ING1 tumor suppressor capable of efficiently killing cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2940. doi:10.1158/1538-7445.AM2015-2940

Abstract B7: Molecular signaling in the human fallopian tube: Insights into proliferation and p53 expression

Abstract Ovarian cancer is the most lethal gynecological malignancy in women. This is primarily due to patients presenting in late stage disease when the cancer has already metastasized. Contributing to this delayed diagnosis is uncertainty regarding the origin of ovarian cancer and what causes initial transformation. It has recently been hypothesized that the distal epithelia of the fallopian tube is the origin of transformation leading to ‘ovarian’ cancer, particularly the serous histotype. A potential precursor lesion to serous cancer, known as the ‘p53 signature’, has been identified in the fallopian tube. However, little is known about the normal human fallopian tube and what factor(s) might lead this potential precursor lesion to develop into serous ovarian cancer. In order to examine the role of the fallopian tube in the development of serous cancer, as well as potential initiation factors that might be involved in early disease, a novel three-dimensional (3D) culture method was utilized. This 3D culture supports growth within the normal tissue architecture and retains epithelia in contact with its stromal environment using alginate hydrogels. This system was utilized for examination of normal human fallopian tissue collected from women undergoing salpingectomy for both benign and malignant conditions. Donated tissues were treated with factors hypothesized to have a role in the onset of serous cancer for 2 and 7 days in culture. Insulin (5 ug/ml) was used to stimulate glycolysis, hydrogen peroxide (H2O2, 1 mM) was used as an oxidative stress mimetic, and the dominant female steroid hormones estrogen (10 nM) and progesterone (100 nM) were utilized to investigate hormone controlled cell growth. Resulting cultured tissues retained normal fallopian morphology as demonstrated by immunohistochemistry for cytokeratin-8 and E-cadherin. Additionally, both tubal epithelial sub-types (secretory and ciliated) were retained in culture (denoted by PAX8 and acetylated-tubulin staining, respectively). Fimbriae cultures were further analyzed for changes in proliferation, DNA damage, and importantly, induction of p53 expression, which is thought to be linked to the onset of serous cancer. Initial findings reveal ex vivo inducible p53 expression can be acquired. These hotspots of p53 expression are secretory cell enriched similarly to those seen in vivo, but are not always expressed concomitantly with γH2AX, a marker of DNA damage. Additionally, it was noted that oxidative stress (H2O2) and insulin significantly increased the rate of proliferation as compared to vehicle control. Conversely, the effect of the steroid hormones on proliferation was less notable, with progesterone treatment consistent with possible promotion of fallopian epithelial cell death. Characterization of biological function within early putative preneoplastic lesions is integral to ovarian cancer research. Without identifying the tissue of origin, current research may be focusing on genetic or protein changes that are different only because they are compared to the wrong normal tissue of origin. Further, targeted therapies might fail due to identification of targets that are not in the correct progenitor cell population. In order to evaluate the p53 signature as a potential precursor lesion to serous cancer, we must understand what induces p53 expression in the fallopian tube and why. Citation Format: Sharon L. Eddie, Suzanne M. Quartuccio, Jessica A. Shepherd, Rajul Kothari, Joanna E. Burdette. Molecular signaling in the human fallopian tube: Insights into proliferation and p53 expression. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B7.

P53-dependent chemokine production by senescent tumor cells supports NKG2D-dependent tumor elimination by natural killer cells

The induction of cellular senescence is an important mechanism by which p53 suppresses tumorigenesis. Using a mouse model of liver carcinoma, where cellular senescence is triggered in vivo by inducible p53 expression, we demonstrated that NK cells participate in the elimination of senescent tumors. The elimination of senescent tumor cells is dependent on NKG2D. Interestingly, p53 restoration neither increases ligand expression nor increases the sensitivity to lysis by NK cells. Instead, p53 restoration caused tumor cells to secrete various chemokines with the potential to recruit NK cells. Antibody-mediated neutralization of CCL2, but not CCL3, CCL4 or CCL5, prevented NK cell recruitment to the senescent tumors and reduced their elimination. Our findings suggest that elimination of senescent tumors by NK cells occurs as a result of the cooperation of signals associated with p53 expression or senescence, which regulate NK cell recruitment, and other signals that induce NKG2D ligand expression on tumor cells.

Docetaxel and 5-fluorouracil induce human p53 tumor suppressor gene transcription via a short sequence at core promoter element

The p53 tumor suppressor protein is involved in cellular defense against agents that can cause genetic damage. Induction of p53 gene expression at transcriptional and post-transcriptional levels by such agents results in p53-regulated gene activation or suppression. Docetaxel (DOC), a member of the taxanes family that is widely used in cancer chemotherapy, activates p53 at the transcriptional level. We demonstrated that p53 is induced by low dose DOC treatment, resulting in MDR-1 gene suppression in human lung cancer cells. To identify the cis-element of p53 promoter that responds to DOC, p53 promoter region was cloned and promoter activity was analyzed on luciferase gene reporter assay. Promoter region (−78 to +129) contained the highest basal p53 promoter activity and deletion of +86 to +129 severely reduced basal promoter activity. Basal promoter region included the 21-bp element (PE21) that determines UV-inducible expression of p53 and mediates DOC-inducible p53 expression. On site-specific mutagenesis of PE21 (−78 to −58), with mutation of ATTG (−62 to −59) to CGGT, completely diminished the response to DOC. The same mutations also inhibited 5-fluorouracil (5-FU)-inducible p53 expression. Our data revealed that a sequence located at PE21 of p53 core promoter regulates p53 induction by chemotherapeutic agents.

The Effects of G-CSF on Proliferation of Mouse Myocardial Microvascular Endothelial Cells

This paper explores the effect of granulocyte colony-stimulating factor (G-CSF) on mouse myocardial microvascular endothelial cell (CMECs) proliferation. CMECs were harvested from C57/BL6 mice. CMECs were cultured in medium containing G-CSF (0 ng/mL, 20 ng/mL, 40 ng/mL, 60 ng/mL) for five days. Proliferative activity of CMECs was examined by CCK-8 method. Hypoxia inducible factor-1 (HIF-1) and p53 expression levels was determined from the mRNA obtained by reverse transcription polymerase chain reaction (RT-PCR). Results showed that the purity quotient of the CMECs, which were cultured by the method of modified myocardial tissue explant culture, was higher than 95%. Compared with control untreated cells, the proliferative activity of CMECs and the expression level of HIF-1 mRNA in these cells were enhanced by G-CSF treatment, whereas the expression level of p53 mRNA was markedly reduced. It may be concluded that G-CSF could promote the proliferative activity of CMECs, which might be mediated by upregulation of HIF-1 and downregulation of p53.

Different levels of p53 induced either apoptosis or cell cycle arrest in a doxycycline-regulated hepatocellular carcinoma cell line in vitro

Induction of p53 gene expression in cancer cells can lead to both cell cycle arrest and apoptosis. To clarify whether the level of p53 expression determines the apoptotic response of hepatocellular carcinoma (HCC) cells, we assessed the effect of various levels of expression of p53 gene on a p53-deficient HCC cell line, Hep3B, utilizing a doxycycline (Dox)-regulated inducible p53 expression system. Our results showed that apoptosis was induced in HCC cells with high levels of p53 expression. However, lower level of p53 expression induced only cell cycle arrest but not apoptosis. Bax expression was up-regulated following high levels of p53 expression, while bcl-2 expression was not altered by the level of p53 expression. Moreover, p21 expression was observed in both high and low expression of p53. These results suggest the level of p53 expression could determine if the HCC cells would go into cell cycle arrest or apoptosis. Bax may participate, at least in part, in inducing p53-dependent apoptosis and the induction of p21 alone was able to cause cell cycle arrest but not apoptosis.

Effect of inducible FHIT and p53 expression in the Calu-1 lung cancer cell line

Loss of FHIT expression and p53 mutations are critical events in the early stages of lung carcinogenesis. The restoration of Fhit function in FHIT-negative cancer cells has been reported to cause tumour suppression by inhibition of cell proliferation and/or activation of apoptotic pathways. However, the studies designed to elucidate the biological role of Fhit and its potential interaction with p53 have produced conflicting results. We investigated here the effects of the simultaneous restoration of FHIT and p53 in Calu-1 cells by using a hormone-inducible gene expression system. We demonstrate that the restoration of FHIT expression reinforces the anti-proliferative effect associated with the simultaneous replacement of p53. Indeed, a more pronounced inhibition of cell proliferation associated with an earlier and higher induction of p21 waf1 mRNA and protein expression was observed in Fhit/p53-expressing cells compared with cells expressing p53 alone. This effect was not due to Fhit-mediated up-regulation of p53 expression; in fact p53 protein was expressed at the same level in both FHIT-positive and FHIT-negative cell clones. Consistent with this result, Fhit did not affect the expression of MDM2, a protein known to interact directly with p53 and target p53 for proteolytic degradation, thus down-regulating its activity.

P-943 Effect of inducible Fhit and p53 expression in Calu-1 lung cancercell line

P-943 Effect of inducible Fhit and p53 expression in Calu-1 lung cancercell line

P53 but not p16INK4a induces growth arrest in retinoblastoma-deficient hepatocellular carcinoma cells

Background/Aim: Both p16INK4a and p53 proteins are negative regulators of the cell cycle. In human hepatocellular carcinomas (HCC), the loss of function of p53, retinoblastoma (pRb) and p16INK4a genes by different mechanisms has been largely documented, but their hepatocellular effects are poorly known. We compared the growth-inhibitory effects of p16INK4a and p53 proteins in Hep3B cell line-derived clones.Methods: Cells were transfected with inducible p16INK4a and p53 expression vectors, and stable clones were analyzed for transgene expression by Western blotting and immunoperoxidase staining. Effects on cell growth were analyzed by in vitro growth assay, thymidine incorporation and flow cytometry. Biochemical effects of p53 were tested by Northern blotting of p21Cip1 transcripts and by Western blotting of p21Cip1, mdm-2, bax, cyclin-dependent kinase 2 and cyclin E proteins. The pRb protein was studied by Western blotting and immunoprecipitation assays.Results: The induction of p16INK4a protein expression did not affect in vitro growth of cells. In contrast, p53 protein in its wild-type conformation provoked a growth arrest accompanied by transactivation of p21Cip1 gene and accumulation of p21Cip1, bax and mdm-2 proteins. p53-induced growth arrest was due to a cell cycle arrest at the G1/S transition, probably mediated by p21Cip1 protein, which inhibits cyclin-dependent kinase 2/cyclin E complexes.Conclusions: The lack of detectable pRb protein and resistance of cells to p16INK4a strongly suggest that p53 is able to arrest the growth of HCC cells by a mechanism independent of“p53-retinoblastoma pathway”. These findings are applicable to HCC with abberrations of both p53 and pRb genes, and may not represent the universal effects of p53 in hepatic cells.

Phenotypic alterations of small cell lung carcinoma induced by different levels of wild-type p53 expression.

p53 induces both growth arrest and apoptosis in cancer cells. To clarify whether the level of p53 expression determines the response of small cell lung carcinoma (SCLC) cells, we assessed the effect of various p53 levels on a p53-null SCLC cell line, N417, using a tetracycline (Tc)-regulated inducible p53 expression system. Apoptosis was induced in SCLC cells with high p53 expression. Although low levels of p53 induced G1 arrest accompanied by p21 expression, cells with G1 arrest seemed to undergo apoptosis after further cultivation. Expression of exogenous p21 induced G1 arrest but not apoptosis in SCLC cells, suggesting that p53-mediated G1 arrest was induced through p21 expression. Moreover, high level of p53 expression down-regulated Bcl-2 expression in SCLC cells, while Bax was consistently expressed irrespective to the level of p53 expression. These results suggest that p53-mediated apoptosis and G1 arrest depend on level of p53 expression in SCLC cells and that the relative dominancy of Bax to Bcl-2 is involved in the induction of apoptosis by high level of p53 expression.

Wild-type p53 triggers a rapid senescence program in human tumor cells lacking functional p53.

The p53 tumor suppressor gene has been shown to play an important role in determining cell fate. Overexpression of wild-type p53 in tumor cells has been shown to lead to growth arrest or apoptosis. Previous studies in fibroblasts have provided indirect evidence for a link between p53 and senescence. Here we show, using an inducible p53 expression system, that wild-type p53 overexpression in EJ bladder carcinoma cells, which have lost functional p53, triggers the rapid onset of G1 and G2/M growth arrest associated with p21 up-regulation and repression of mitotic cyclins (cyclin A and B) and cdc2. Growth arrest in response to p53 induction became irreversible within 48-72 h, with cells exhibiting morphological features as well as specific biochemical and ultrastructural markers of the senescent phenotype. These findings provide direct evidence that p53 overexpression can activate the rapid onset of senescence in tumor cells.