Wt P53 Protein Research Articles

Selective Targeting of TP53-Y220C Mutant AML By PC14586 Results in TP53 Wild-Type Conformation and Synergistical Apoptosis Induction By Concomitant Inhibition of Xpo-1, MDM2, or Bcl-2

Patients with TP53-mutant acute myeloid leukemia (AML) constitute the worst outcome group with a survival of only several months. TP53 mutations frequently become dominant following exposure to chemotherapeutic agents, Bcl-2 or MDM2 inhibitors, and confer resistance to multiple other therapies. Y220C is a recurrent hotspot TP53 mutation observed in solid tumors and hematological malignancies, predominantly in AML and MDS (Gener-Ricoset al., ASH 2022). PC14586 (PMV Pharmaceuticals) is designed to bind to the p53-Y220C mutant and restore wild-type (WT) p53 protein conformation and function. PC14586 has been shown to convert Y220C-p53 mutant into WT-p53 protein conformation, induce the activation of p53 transcriptional pathways resulting in selective inhibition of proliferation ofTP53-Y220C mutant solid tumor cell lines and achieved partial responses and stable disease in an ongoing phase I clinical trial in solid tumors (Dumbrava, ASCO 2022, NCT04585750). However, the activity of PC14586 in TP53-Y220C mutated AML/MDS has not been investigated, either as monotherapy or in mechanism-based drug combinations. We investigated the mechanisms of action of PC14586 in AML using cells with TP53-WT, knockout (KO), and mutations (one allele with Y220C or R175H and the other truncated) generated by CRISPR (Boettcher et al., Science 2019) and found that PC14586 converts Y220C mutant p53 to the active form of p53 and selectively activates p53 signaling proteins including MDM2 and p21 in TP53-Y220C AML cells. PC14586 decreased cell viability but had limited apoptogenic activities in the TP53-Y220C AML cells . In primary AML cells with Y220C mutations, PC14586 induced cell death in bulk AML cells and, importantly, also in stem/progenitor cells in 2/3 samples. We reported previously that sustained nuclear retention of p53 by XPO-1 inhibition greatly enhances the transcriptional activity of activated p53 and that activation of p53 by inhibiting its negative regulator MDM2 effectively induces cell death in TP53-WT AML (Kojima et al., Blood 2013, 2005), and that XPO-1 inhibition vastly increased apoptosis induced by MDM2 inhibition through 20-60-fold increased p53 transcriptional activities. We also reported that combined Bcl-2 inhibition and p53 activation is synthetically lethal in TP53-WT AML (Pan et al., Cancer Cell 2017). We hypothesized that XPO-1 synergizes with PC14586 by retaining PC14586-activated p53 in the nucleus and that inhibition of MDM2 induced by PC14586-mediated p53 activation or inhibition of Bcl-2 synergizes with PC14586 in TP53-Y220C AML cells. Indeed, we found that PC14586-induced p53 target proteins were further upregulated when PC14586 was combined with XPO-1 or MDM2 inhibition. Inhibition of XPO-1, MDM2, or Bcl-2 synergistically induced massive apoptosis and decreased cell viability in PC14586 treated TP53 Y220C mutant AML cells (Fig.1), in primary AML blasts, and in CD34+CD38- stem/progenitor cells but not in normal bone marrow cells or stem/progenitor cells through conformational changes of mutant p53 to p53-WT. While PC14586 had limited activity on proliferation in TP53-Y220C AML cells, the combination with Bcl-2 inhibition largely blocked G1-S transition and enhanced apoptosis. The G1-S transition block and apoptosis induction were even more pronounced when PC14586 was combined with XPO-1 or MDM2 inhibitors as determined by flow cytometry measurement of DNA content, and PARP cleavage. Experiments in TP53-Y220C AML PDX models are ongoing and will be reported. In summary, PC14586 converts Y220C-mutant p53 into WT-p53 conformation and induces p53 target proteins. Mechanism-based combinations with XPO-1, MDM2, and Bcl-2 inhibitors induce massive apoptosis. PC14586 is presently in early clinical trials.

Transcriptional profiling reveals a subset of human breast tumors that retain wt TP53 but display mutant p53-associated features.

TP53 gene mutations are very common in human cancer. While such mutations abrogate the tumor suppressive activities of the wild‐type (wt) p53 protein, some of them also endow the mutant (mut) protein with oncogenic gain of function (GOF), facilitating cancer progression. Yet, p53 may acquire altered functionality even without being mutated; in particular, experiments with cultured cells revealed that wtp53 can be rewired to adopt mut‐like features in response to growth factors or cancer‐mimicking genetic manipulations. To assess whether such rewiring also occurs in human tumors, we interrogated gene expression profiles and pathway deregulation patterns in the METABRIC breast cancer (BC) dataset as a function of TP53 gene mutation status. Harnessing the power of machine learning, we optimized a gene expression classifier for ER+Her2‐ patients that distinguishes tumors carrying TP53 mutations from those retaining wt TP53. Interestingly, a small subset of wt TP53 tumors displayed gene expression and pathway deregulation patterns markedly similar to those of TP53‐mutated tumors. Moreover, similar to TP53‐mutated tumors, these ‘pseudomutant’ cases displayed a signature for enhanced proliferation and had worse prognosis than typical wtp53 tumors. Notably, these tumors revealed upregulation of genes which, in BC cell lines, were reported to be positively regulated by p53 GOF mutants. Thus, such tumors may benefit from mut p53‐associated activities without having to accrue TP53 mutations.

Pentamidine niosomes thwart S100B effects in human colon carcinoma biopsies favouring wtp53 rescue.

S100B protein bridges chronic mucosal inflammation and colorectal cancer given its ability to activate NF‐kappaB transcription via RAGE signalling and sequestrate pro‐apoptotic wtp53. Being an S100B inhibitor, pentamidine antagonizes S100B‐wtp53 interaction, restoring wtp53‐mediated pro‐apoptotic control in cancer cells in several types of tumours. The expression of S100B, pro‐inflammatory molecules and wtp53 protein was evaluated in human biopsies deriving from controls, ulcerative colitis and colon cancer patients at baseline (a) and (b) following S100B targeting with niosomal PENtamidine VEhiculation (PENVE), to maximize drug permeabilization in the tissue. Cultured biopsies underwent immunoblot, EMSA, ELISA and biochemical assays for S100B and related pro‐inflammatory/pro‐apoptotic proteins. Exogenous S100B (0.005‐5 μmol/L) alone, or in the presence of PENVE (0.005‐5 μmol/L), was tested in control biopsies while PENVE (5 μmol/L) was evaluated on control, peritumoral, ulcerative colitis and colon cancer biopsies. Our data show that S100B level progressively increases in control, peritumoral, ulcerative colitis and colon cancer enabling a pro‐inflammatory/angiogenic and antiapoptotic environment, featured by iNOS, VEGF and IL‐6 up‐regulation and wtp53 and Bax inhibition. PENVE inhibited S100B activity, reducing its capability to activate RAGE/phosphor‐p38 MAPK/NF‐kappaB and favouring its disengagement with wtp53. PENVE blocks S100B activity and rescues wtp53 expression determining pro‐apoptotic control in colon cancer, suggesting pentamidine as a potential anticancer drug.

Targeting Misfolded p53 and p53 Aggregation to Overcome Resistance to Apoptosis in Acute Myeloid Leukemia

Targeting Misfolded p53 and p53 Aggregation to Overcome Resistance to Apoptosis in Acute Myeloid Leukemia

The pharmalogical reactivation of p53 function improves breast tumor cell lysis by granzyme B and NK cells through induction of autophagy

Cytotoxic T lymphocytes (CTL) and natural killer cells (NK)-mediated elimination of tumor cells is mostly dependent on Granzyme B apoptotic pathway, which is regulated by the wild type (wt) p53 protein. Because TP53 inactivating mutations, frequently found in human tumors, could interfere with Granzyme B-mediated cell death, the use of small molecules developed to reactivate wtp53 function in p53-mutated tumor cells could optimize their lysis by CTL or NK cells. Here, we show that the pharmalogical reactivation of a wt-like p53 function in p53-mutated breast cancer cells using the small molecule CP-31398 increases their sensitivity to NK-mediated lysis. This potentiation is dependent on p53-mediated induction of autophagy via the sestrin-AMPK-mTOR pathway and the ULK axis. This CP31398-induced autophagy sequestrates in autophagosomes several anti-apoptotic proteins, including Bcl-XL and XIAP, facilitating Granzyme B-mediated mitochondrial outer membrane permeabilization, caspase-3 activation and Granzyme B- or NK cell-induced apoptosis. Together, our results define a new way to increase cytotoxic lymphocyte-mediated lysis of p53-mutated breast cancer cell, through a p53-dependent autophagy induction, with potential applications in combined immunotherapeutic approaches.

Abstract SY32-02: Modulation of wild-type p53 activity in cancer cells and in the tumor microenvironment

Abstract While the TP53 gene is mutated in about half of all human cancer cases, it remains wild type (wt) in the other half, suggesting that its tumor suppressor activities are either abrogated or circumvented in the latter cases. Moreover, while early evidence indicates that p53 can also exert non-cell-autonomous tumor suppressive activities in the tumor stroma, p53 typically remains wt in stromal cells. These observations raise the question of what exactly happens to p53 in tumors where the TP53 gene is not mutated. The two main alternative scenarios that come to mind are: 1. The protein is retained in functional state, but is unable to exert measurable biological effects in particular contexts or particular cell types; 2. The protein loses its functionality, either because it is not expressed at all (e.g. the gene is silenced or the protein is degraded), or because it is subjected to inhibitory molecular processes (e.g. protein-protein interactions or post-translational modifications) that deprive it of its functionality. In both scenarios, further depletion of the endogenous wtp53 is not expected to have significant biological consequences. Recent work from our lab has provided support for a third scenario, wherein the p53 protein is wt by sequence, but nevertheless acquires properties that are usually ascribed to cancer-associated mutant p53 isoforms, namely oncogenic gain-of-function properties. This has been demonstrated in human mammary epithelial cells in which key components of the Hippo tumor suppressor pathway have been silenced, as well as in cancer-associated fibroblasts. In both situations, depletion of the endogenous wtp53 protein leads to attenuation of features associated with cancer aggressiveness, such as cancer cell migration and invasion, implying that the wtp53 of those cells actually contributes to a more aggressive cancer phenotype. We thus proposed that such “oncogenic rewiring” converts wtp53 from tumor suppressor to cancer promoter. In an attempt to evaluate whether rewiring of wtp53 can be observed in actual human tumors, we interrogated gene expression profiles and pathway deregulation patterns in the METABRIC breast cancer dataset as a function of TP53 mutation status (subsequently validated also on the TCGA dataset). As expected, a gene expression signature that distinguishes between tumors that carry TP53 mutations and those that retain wt TP53 could be defined and validated. Interestingly, a small subset of wt TP53 tumors nevertheless displayed gene expression and pathway deregulation patterns that were markedly similar to those observed in TP53-mutated tumors, and distinct from the patterns that are characteristic of the bulk of wt TP53 breast tumors. Conceivably, this subset of tumors may represent situations where the p53 protein is not expressed or is rapidly eliminated. However, at least in several of those cases, p53 expression was observable by immunohistochemistry, suggesting that the wtp53 protein is rewired rather than absent. Analysis of the transcriptional profile of this “pseudomutant” subset of wt TP53 tumors identified a subset of pathways that are preferentially deregulated in those tumors as compared to the other wtp53 tumors and the TP53-mutated tumors, raising the possibility that their deregulation may play a role in the rewiring of wt p53 into a “pseudomutant” state. Initial experiments aimed to address this possibility in human breast cancer cells will be presented. Citation Format: Moshe Oren, Sharathchandra Arandkar, Gal Benor, Noa Furth, Yael Aylon, Eytan Domany. Modulation of wild-type p53 activity in cancer cells and in the tumor microenvironment [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 SY32-02.

Wild-type p53 oligomerizes more efficiently than p53 hot-spot mutants and overcomes mutant p53 gain-of-function via a “dominant-positive” mechanism

Human p53 protein acts as a transcription factor predominantly in a tetrameric form. Single residue changes, caused by hot-spot mutations of the TP53 gene in human cancer, transform wild-type (wt) p53 tumor suppressor proteins into potent oncoproteins - with gain-of-function, tumor-promoting activity. Oligomerization of p53 allows for a direct interplay between wt and mutant p53 proteins if both are present in the same cells – where a mutant p53's dominant-negative effect known to inactivate wt p53, co-exists with an opposite mechanism – a “dominant-positive” suppression of the mutant p53's gain-of-function activity by wt p53. In this study we determine the oligomerization efficiency of wt and mutant p53 in living cells using FRET-based assays and describe wt p53 to be more efficient than mutant p53 in entering p53 oligomers. The biased p53 oligomerization helps to interpret earlier reports of a low efficiency of the wt p53 inactivation via the dominant-negative effect, while it also implies that the “dominant-positive” effect may be more pronounced. Indeed, we show that at similar wt:mutant p53 concentrations in cells – the mutant p53 gain-of-function stimulation of gene transcription and cell migration is more efficiently inhibited than the wt p53's tumor-suppressive transactivation and suppression of cell migration. These results suggest that the frequent mutant p53 accumulation in human tumor cells does not only directly strengthen its gain-of-function activity, but also protects the oncogenic p53 mutants from the functional dominance of wt p53.

Mutations of p53 decrease sensitivity to the anthracycline treatments in bladder cancer cells.

Due to doxorubicin (Dox) cardiotoxicity, the next generation of novel non-cardiotoxic anthracyclines, including AD 312 and AD 198, were synthesized and validated. In this study, we assessed the efficacy and mechanisms of anthracyclines-induced apoptosis and inhibition of cell viability in human bladder cancer cells expressing wild-type (wt) p53 (RT4 and SW780) and mutated (mt) p53 (UM-UC-3, 5637, T-24, J82, and TCCSUP) protein. Anthracyclines inhibited cell viability in tested TCC cells, but were less effective in mt-p53 TCC cells, especially in the drug-resistant J82 and TCCSUP cells. Anthracyclines upregulated the expression of wt p53 protein in RT4 and SW780 cells, but had no effect on expression of mt p53 protein in UM-UC-3, 5637, T-24, J82, and TCCSUP cells. The anthracyclines activated caspase 3/7 and cleavage of PARP in wt-p53 RT4 and SW780 cells, and mt-p53 5637, UM-UC-3, and T-24, but not in mt-p53 J82 and TCCSUP cells. The anthracyclines-induced cleavage of PARP was blocked by p53 siRNA in wt-p53 RT4 cells. Co-treatment of AD 198 with PRIMA-1 significantly inhibited cell viability of mt-p53 J82 cells, but had no effect in wt-p53 RT4 cells. AD 198 blocked c-myc expression in mt-p53 UM-UC-3, 5637, T-24, and J82 cells, however no expression of c-myc was detected in wt-p53 RT4 and SW780 cells. In conclusion, our results demonstrated that the anthracycline-induced resistance in bladder cancer cells positively correlated with TP53 mutations in the tetramerization domain in J82 and TCCSUP cells. Further, AD 312 and AD 198 are promising chemotherapeutic drugs for bladder cancer, especially in combination with PRIMA-1.

A Combination of SAHA and Quinacrine Is Effective in Inducing Cancer Cell Death in Upper Gastrointestinal Cancers.

Purpose: We aimed to investigate the therapeutic efficacy of single agent and the combination of quinacrine and suberoylanilide hydroxamic acid (SAHA) in wt- and mut-p53 upper gastrointestinal cancer (UGC) cell models.Experimental Design: ATP-Glo, clonogenic cell survival, Annexin V, comet, DNA double-strand breaks (DSBs), qPCR, and Western blot analysis assays were utilized.Results: Using clonogenic cell survival, ATP-Glo cell viability, Annexin V, and sub-G0 population analysis, we demonstrated that a combination of quinacrine and SAHA significantly decreased colony formation and increased cancer cell death (range, 4-20 fold) in six UGC cell models, as compared with single-agent treatments, irrespective of the p53 status (P < 0.01). The combination of quinacrine and SAHA induced high levels of DSB DNA damage (>20-fold, P < 0.01). Western blot analysis showed activation of caspases-3, 9, and γ-H2AX in all cell models. Of note, although quinacrine treatment induced expression of wt-p53 protein, the combination of quinacrine and SAHA substantially decreased the levels of both wt-P53 and mut-P53. Furthermore, cell models that were resistant to cisplatin (CDDP) or gefitinib treatments were sensitive to this combination. Tumor xenograft data confirmed that a combination of quinacrine and SAHA is more effective than a single-agent treatment in abrogating tumor growth in vivo (P < 0.01).Conclusions: Our novel findings show that the combination of quinacrine and SAHA promotes DNA damage and is effective in inducing cancer cell death, irrespective of p53 status and resistance to CDDP or gefitinib in UGC models. Clin Cancer Res; 24(8); 1905-16. ©2018 AACR.

The First Approved Gene Therapy Product for Cancer Ad-p53 (Gendicine): 12 Years in the Clinic.

Gendicine (recombinant human p53 adenovirus), developed by Shenzhen SiBiono GeneTech Co. Ltd., was approved in 2003 by the China Food and Drug Administration (CFDA) as a first-in-class gene therapy product to treat head and neck cancer, and entered the commercial market in 2004. Gendicine is a biological therapy that is delivered via minimally invasive intratumoral injection, as well as by intracavity or intravascular infusion. The wild-type (wt) p53 protein expressed by Gendicine-transduced cells is a tumor suppressor that is activated by cellular stress, and mediates cell-cycle arrest and DNA repair, or induces apoptosis, senescence, and/or autophagy, depending upon cellular stress conditions. Based on 12 years of commercial use in >30,000 patients, and >30 published clinical studies, Gendicine has exhibited an exemplary safety record, and when combined with chemotherapy and radiotherapy has demonstrated significantly higher response rates than for standard therapies alone. In addition to head and neck cancer, Gendicine has been successfully applied to treat various other cancer types and different stages of disease. Thirteen published studies that include long-term survival data showed that Gendicine combination regimens yield progression-free survival times that are significantly longer than standard therapies alone. Although the p53 gene is mutated in >50% of all human cancers, p53 mutation status did not significantly influence efficacy outcomes and long-term survival rate for Ad-p53-treated patients. To date, Shenzhen SiBiono GeneTech has manufactured 41 batches of Gendicine in compliance with CFDA QC/QA requirements, and 169,571 vials (1.0 × 1012 vector particles per vial) have been used to treat patients. No serious adverse events have been reported, except for vector-associated transient fever, which occurred in 50-60% of patients and persisted for only a few hours. The manufacturing accomplishments and clinical experience with Gendicine, as well as the understanding of its cellular mechanisms of action and implications, could provide valuable insights for the international gene therapy community and add valuable data to promote further developments and advancements in the gene therapy field.

Abstract 1095: A combination of suberoylanilide hydroxamic acid and quinacrine is an effective therapeutic approach in preclinical settings of upper gastrointestinal cancers

Abstract Background: Quinacrine (QC), an antimalarial drug, has been shown to possess anticancer effects. Suberoylanilide hydroxamic acid (SAHA) inhibits class I and class II HDACs and is approved for cancer therapy. Developing novel approaches to overcome cancer drug resistance could significantly enhance current therapeutic approaches and improve patient care. Methods: ATP-GLO, clonogenic survival, Annexin-V apoptosis assay, comet assay and DNA double-strand breaks (DSB) kits were used. The mRNA and protein levels were evaluated by quantitative real-time PCR and Western blot analyses. Results: A combination of QC/SAHA significantly increased cell death in all cancer cell lines and had no effect on immortalized non-cancer cell lines (HFE145, NIH-3T3 and EPC2) (P&amp;lt;0.01). Clonogenic survival assay indicated that QC/SAHA co-treatment led to significantly lower number of cancer cell colonies, as compared to single agents and controls (P&amp;lt;0.01). Of note, the QC/SAHA combination led to an increase in the the sub-G0 population in AGS (9-fold), MKN-28 (14-fold), FLO1 (5.6-fold), and SNU1 (4-fold) cells (P&amp;lt;0.01). These results were confirmed using the Annexin V apoptosis induced significantly higher levels of apoptosis (10 - 20 fold) as compared to single agent and control (P&amp;lt;0.01). Treatment with QC/SAHA combination induced high levels of DSB (&amp;gt;20 fold, P&amp;lt;0.01). Comet assay data showed increased DNA damage compared with vehicle-treated cells (8-fold, P&amp;lt;0.01). Western blot analysis demonstrated a notable increase in activation of PARP, caspases 3, 9 and γ-H2AX following QC/SAHA co-treatment in all cancer cell lines. Interestingly, the combination of QC/SAHA substantially decreased the protein levels of both wtP53 and mutP53 in these cells. Tumor xenograft data confirmed that a combination of QC/SAHA is more effective than a single agent in abrogating tumor growth (P&amp;lt;0.05). Conclusion: Our novel findings show that QC and SAHA have a synergistic effect on cancer cell death. The results provide compelling evidence that increased DNA damage mediates the cytotoxic effect of combined QC/SAHA. Such effect is likely related to mutP53 and wtP53 protein degradation induced by QC/SAHA combination. Our findings provide a rationale for a clinical evaluation of combined QC/SAHA therapy in gastro-esophageal cancers. Ongoing studies are currently being undertaken to understand mechanisms that regulate the degradation of wtp53 and mutp53 proteins. Citation Format: Shoumin Zhu, Wael El-Rifai. A combination of suberoylanilide hydroxamic acid and quinacrine is an effective therapeutic approach in preclinical settings of upper gastrointestinal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1095. doi:10.1158/1538-7445.AM2017-1095

Cooperation of Musashi-2, Numb, MDM2, and P53 in drug resistance and malignant biology of pancreatic cancer.

Our earlier work showed that Musashi (MSI)-2 promoted the development of pancreatic cancer (PC) by down-regulating Numb, which prevented murine double-minute (MDM)-2-mediated p53 ubiquitin degradation. Thus, we investigate the relationship among MSI2, Numb, MDM2, and p53 in PC in vitro and invivo, an association that has not been reported to our knowledge. MSI2 had no relationship with mutant p53 (mtp53) and wild-type p53 (wtp53) in normal PC cells. However, in response to gemcitabine or cisplatin treatment, MSI2 silencing simultaneously down-regulated MDM2 and up-regulated Numb and wtp53 protein levels. Moreover, these 4 endogenous proteins can be coimmunoprecipitated as a quaternary complex. Numb small interfering RNA (siRNA) reversed the MSI2 silencing-induced p53 increase. During treatment with chemical agents, MSI2 silencing decreased drug resistance and cell motility in vitro and inhibited tumor growth in vivo, all of which were significantly reversed by p53 siRNA. MSI2 was also negatively associated with Numb and positively associated with MDM2 expression in tissue. Overexpression of MSI2, MDM2, and mtp53 and weak expression of Numb were closely associated with aggressive clinicopathologic characteristics and poor prognosis for patients with PC. MSI2 negatively regulates wtp53 protein by up-regulating MDM2 and down-regulating Numb after treatment with chemical agents. MSI2 promotes drug resistance and malignant biology of PC in a p53-dependent manner.-Sheng, W., Dong, M., Chen, C., Wang, Z., Li, Y., Wang, K., Li, Y., Zhou, J. Cooperation of Musashi-2, Numb, MDM2, and P53 in drug resistance and malignant biology of pancreatic cancer.

Low-dose irradiation promotes proliferation of the human breast cancer MDA-MB-231 cells through accumulation of mutant P53.

Low-dose irradiation (LDIR) has been proven to have differential biological effects on normal mammalian somatic cells and cancer cells. Our previous study showed that p53 gene status is a critical factor regulating the effect of LDIR on cancer cells. We investigated the effect of LDIR on the breast cancer cell line MDA-MB-231 that harbors a mutant p53 gene, and the normal breast fibroblast cell line Hs 578Bst. In the present study, we showed that 150mGy LDIR pormoted growth of MDA-MB-231 cells but not Hs578Bst cells. Through cell cycle analyses, we found that LDIR accelerated cell cycle into S phase in MDA-MB-231 cells, but did not affect the cell cycle of Hs 578Bst cells. Using western blotting, we demonstrated that the expression of CDK4, CDK6 and cyclinD1 was upregulated in MDA-MB-231 cells after LDIR. Although LDIR increased ataxia-telangiectasia mutated (ATM) level in both MDA-MB-231 cells and Hs578Bst cells and activated ATM/p53/p21 pathway, only the mutant type of p53 (mtp53) protein in MDA-MB-231 cells was shown to be accumulated after LDIR. Using ATM inhibitor or lentivirus-mediated small interfering RNA (siRNA) to block the ATM/p53/p21 pathway in MDA-MB-231 cells, the LDIR-induced cell proliferation was abolished. When we introduced wild-type p53 (wtp53) protein into MDA-MB-231 cells, the LDIR-induced cell proliferation was also abolished. These findings suggest that normal p53 function is crucial in ATM/p53/p21 pathway activated by LDIR. The p53 status is the most probable reason leading to differential LDIR biological activities between breast tumor cells and normal breast cells.

Oncogenic Intra-p53 Family Member Interactions in Human Cancers.

The p53 gene family members p53, p73, and p63 display several isoforms derived from the presence of internal promoters and alternative splicing events. They are structural homologs but hold peculiar functional properties. p53, p73, and p63 are tumor suppressor genes that promote differentiation, senescence, and apoptosis. p53, unlike p73 and p63, is frequently mutated in cancer often displaying oncogenic “gain of function” activities correlated with the induction of proliferation, invasion, chemoresistance, and genomic instability in cancer cells. These oncogenic functions are promoted either by the aberrant transcriptional cooperation of mutant p53 (mutp53) with transcription cofactors (e.g., NF-Y, E2F1, Vitamin D Receptor, Ets-1, NF-kB and YAP) or by the interaction with the p53 family members, p73 and p63, determining their functional inactivation. The instauration of these aberrant transcriptional networks leads to increased cell growth, low activation of DNA damage response pathways (DNA damage response and DNA double-strand breaks response), enhanced invasion, and high chemoresistance to different conventional chemotherapeutic treatments. Several studies have clearly shown that different cancers harboring mutant p53 proteins exhibit a poor prognosis when compared to those carrying wild-type p53 (wt-p53) protein. The interference of mutantp53/p73 and/or mutantp53/p63 interactions, thereby restoring p53, p73, and p63 tumor suppression functions, could be among the potential therapeutic strategies for the treatment of mutant p53 human cancers.

Nitric Oxide Down-Regulates Topoisomerase I and Induces Camptothecin Resistance in Human Breast MCF-7 Tumor Cells.

Camptothecin (CPT), a topoisomerase I poison, is an important drug for the treatment of solid tumors in the clinic. Nitric oxide (·NO), a physiological signaling molecule, is involved in many cellular functions, including cell proliferation, survival and death. We have previously shown that ·NO plays a significant role in the detoxification of etoposide (VP-16), a topoisomerase II poison in vitro and in human melanoma cells. ·NO/·NO-derived species are reported to modulate activity of several important cellular proteins. As topoisomerases contain a number of free sulfhydryl groups which may be targets of ·NO/·NO-derived species, we have investigated the roles of ·NO/·NO-derived species in the stability and activity of topo I. Here we show that ·NO/·NO-derived species induces a significant down-regulation of topoisomerase I protein via the ubiquitin/26S proteasome pathway in human colon (HT-29) and breast (MCF-7) cancer cell lines. Importantly, ·NO treatment induced a significant resistance to CPT only in MCF-7 cells. This resistance to CPT did not result from loss of topoisomerase I activity as there were no differences in topoisomerase I-induced DNA cleavage in vitro or in tumor cells, but resulted from the stabilization/induction of bcl2 protein. This up-regulation of bcl2 protein in MCF-7 cells was wtp53 dependent as pifithrine-α, a small molecule inhibitor of wtp53 function, completely reversed CPT resistance, suggesting that wtp53 and bcl2 proteins played important roles in CPT resistance. Because tumors in vivo are heterogeneous and contaminated by infiltrating macrophages, ·NO-induced down-regulation of topoisomerase I protein combined with bcl2 protein stabilization could render certain tumors highly resistant to CPT and drugs derived from it in the clinic.

Growth inhibitory effect of adenovirus-mediated tissue-targeted expression of ribosomal protein L23 on human colorectal carcinoma cells

A large body of evidence has established murine double minute 2 (MDM2) as a crucial negative regulator of p53 and the major suppressor of p53 function in tumors with wild-type (wt)-p53. Therefore, by inhibiting MDM2 one may reactivate p53 in tumor cells, leading to their demise. Previous studies revealed that ribosomal protein L23 (RPL23) inhibited MDM2-mediated p53 ubiquitination through direct binding to MDM2, and subsequently induced the p53 level as well as its activity, suggesting that it may be a candidate for use in tumor gene therapy. In the present study, we developed a recombinant adenoviral vector expressing the RPL23 gene under control of the carcinoembryonic antigen (CEA) promoter (rAd/CEA-RPL23), and using an in vitro system with cultured human colorectal carcinoma LoVo cells harboring the wt-p53 gene, we proved that rAd/CEA-RPL23 infection could induce the accumulation of endogenous wt-p53 protein and thus lead to the inhibition of tumor cell growth via inducing cell cycle arrest and apoptosis. In vivo treatment of rAd/CEA-RPL23 also exhibited a significant inhibitory effect on tumor growth in nude mice bearing LoVo xenografts. Furthermore, we showed that rAd/CEA-RPL23 synergized with classic chemotherapeutic agent 5-fluorouracil (5-FU) and enhanced its activity against LoVo cells in vivo and in vitro. Taken together, the data presented here suggest that CEA promoter-targeted exogenous RPL23 expression could be of therapeutic value against human colorectal carcinoma that retains wt-p53.

Trans-splicing repair of mutant p53 suppresses the growth of hepatocellular carcinoma cells in vitro and in vivo.

Reactivation of wild-type p53 (wt-p53) function is an attractive therapeutic approach to p53-defective cancers. An ideal p53-based gene therapy should restore wt-p53 production and reduces mutant p53 transcripts simultaneously. In this study, we described an alternative strategy named as trans-splicing that repaired mutant p53 transcripts in hepatocellular carcinoma (HCC) cells. The plasmids which encoded a pre-trans-splicing molecule (PTM) targeting intron 6 of p53 were constructed and then transfected into HCC cells carrying p53 mutation. Phenotypic changes of HCC cells induced by p53-PTM were analyzed through cell cycle, cell apoptosis and the expression of p53 downstream target genes. Spliceosome mediated RNA trans-splicing (SMaRT) reduced mutant p53 transcripts and produced functional wt-p53 protein after the delivery of p53-PTM plasmids, which resulted in phenotype correction of HCC cells. In tumor xenografts established by p53-mutated HCC cells, adenovirus encoding p53-PTM induced cell cycle arrest and apoptosis and then blocked the growth of tumors in mice. Collectively, our results demonstrated for the first time that mutant p53 transcripts were functionally corrected in p53-defective HCC cells and xenografts using trans-splicing, which indicated the feasibility of using trans-splicing to repair p53 mutation in p53-defective cancers.

P53 N-terminal binding and stabilisation by PIAS3 inhibits MDM2-induced p53 ubiquitination and regulates cell growth

Mouse double minute 2 homolog (MDM2) binds to p53 through the 1-52 amino acid region of p53, in order to ubiquitinate p53. MDM2 thus destabilises p53 and inhibits the effect of p53 on cell cycle arrest and apoptosis. In the present study, the 1-52 amino acid region of the wild-type (wt) p53 protein was stably expressed in H1299 cells. The lysate of the transfected cells was then analysed using co-immunoprecipitation. A specific fraction of the precipitate was subjected to mass spectrometry analysis, which revealed that p53 bound to protein inhibitor of activated STAT3 (PIAS3). To the best of our knowledge, the present study is the first to report that the interaction of PIAS3 with p53 occurs through the 1-52 amino acid region of p53. Overexpression of PIAS3 in the A549 wt p53-expressing cell line was found to significantly increase the half‑life of p53 in the presence of cycloheximide, an inhibitor of protein synthesis. However, PIAS3 overexpression did not affect p53 mRNA levels. Furthermore, PIAS3 overexpression was observed to decrease p53 ubiquitination. Protein-protein interaction analysis revealed that PIAS3 binds to the 1-52 amino acid region of p53, thus disrupts the formation of the p53‑MDM2 complex. In addition, overexpression of PIAS3 in A549 cells was found to enhance the transcription of the p53‑transactivated target p21/waf1, due to p53 accumulation, which led to an increase in p53 binding to the p21 gene promoter. These findings indicate that this newly identified p53‑PIAS3 interaction through the 1-52 amino acid region of p53, reduces p53‑MDM2 complex formation, which not only increases the half-life of p53, but also its transactivation of target genes.

Transferrin receptor targeting nanomedicine delivering wild-type p53 gene sensitizes pancreatic cancer to gemcitabine therapy

To overcome gene therapy barriers such as low transfection efficiency and nonspecific delivery, liposomal nanoparticles targeted by a single-chain antibody fragment to the transferrin receptor (TfRscFv) delivering wild-type (wt) human p53 (SGT-53) were developed for tumor-specific targeting. We hypothesize that SGT-53 in combination with gemcitabine will demonstrate enhanced therapeutic benefit in an in vivo metastatic pancreatic cancer model. Intrasplenic injection of 1 × 10(6) Panc02 murine pancreatic cancer cells was used to generate in vivo hepatic metastatic tumors. Nanoparticle localization was assessed by tail vein injection of TfRscFv with fluorescently labeled oligonucleotides (6-carboxyfluorescein phosphoramidite (6FAM) ODN) imaged by Xenogen IVIS 200 scan. SGT-53 (equivalent to 30 μg of p53 intravenously) and gemcitabine (20 mg/kg intraperitoneally) alone and in combination were administered biweekly and compared with untreated mice. Survival was determined by blinded daily assessment of morbidity. Human wtp53 expression and transferrin levels in the tumors were assessed by western blot analysis. Tumor burden was quantified by liver weight. Xenogen imaging demonstrated tumor-specific uptake of TfRscFv-6FAM ODN. Exogenous human wtp53 protein was detected in the SGT-53-treated tumors compared with control. Compared with untreated mice with metastatic tumors demonstrating median survival of 20 days, SGT-53, gemcitabine and the combination demonstrated improved median survival of 29, 30 and 37 days, respectively. The combination treatment prolonged median survival when compared with single drug treatment and decreased tumor burden. The tumor targeting liposomal-based SGT-53 nanoparticle is capable of sensitizing pancreatic cancer to conventional chemotherapy in pancreatic cancer models. This approach has the potential to be translated into a new, more effective therapy for pancreatic cancer. Further optimization is ongoing, moving towards a Phase 1B/2 clinical trial.

Multi-target lentivirus specific to hepatocellular carcinoma: In vitro and in vivo studies

We aimed at investigating the effects of the targeted transduction of the Wtp53-pPRIME-miR30-shRNA gene into liver cancer cells, under the mediation of anti-alpha fetoprotein scFv-directed lentivirus, and the inhibitory effect of this system on liver cancer cells. The result of infection was observed by fluorescence microscopy. Polymerase chain reaction and Western blotting were used to demonstrate the successful transduction and transcription of the Wtp53-pPRIME-miR30-shRNA-IGF1R gene. Cell growth was observed via the Cell-Counting Kit-8 Method, and cell apoptosis was detected by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling. To observe further the effects of AFP-Wtp53-pPRIME-miR30-shRNA-IGF1R therapy in animals, models of BALB-C nude mice bearing subcutaneous human hepatocellular carcinoma were established. The influence of the growth of subcutaneously transplanted tumor, expression of Wtp53 protein, apoptosis, and microvessel formation on the overall level of AFP-Wtp53 pPRIME-miR30-shRNA-IGF1R were also evaluated. Recombinant lentivirus was successfully constructed, and its functional plaque-forming unit titer was determined as 4.58 × 10(9)plaque-forming units/ml. A positive strand was detected by polymerase chain reaction and Western blotting. Lentiviral construction worked effectively in AFP-positive liver cancer cells. In vitro and in vivo experiments showed that the recombinant lentivirus was more efficacious in inhibiting the proliferation of Hep3B cells. The Wtp53-pPRIME-miR30-shRNA gene can be subjected to targeted transduction into liver cancer cells under the mediation of anti-alpha fetoprotein scFv-directed lentivirus. The Wtp53-pPRIME-miR30-shRNA system has targeting ability and lethal effects on liver cancer cells.