Phosphorylation Of P53 Research Articles

The kinesin light chain-2, a target of mRNA stabilizing protein HuR, inhibits p53 protein phosphorylation to promote radioresistance in NSCLC.

Radioresistance hinders radiotherapy for the treatment of lung cancer. Kinesin light chain-2 (KLC2) has been found to be upregulated in lung cancer and also to be associated with poor prognosis. This study aimed to investigate the effect of KLC2 on radiosensitivity in lung cancer. The radioresistant role of KLC2 was determined by colony formation, neutral comet assay, and γH2AX immunofluorescent staining assay. We further verified the function of KLC2 in a xenograft tumor model. The downstream of KLC2 was identified through gene set enrichment analysis and validated by western blot. Finally, we analyzed clinical data from the TCGA database to reveal the upstream transcription factor of KLC2, which was validated by RNA binding protein immunoprecipitation assay. Here, we found that downregulation of KLC2 could significantly reduce colony formation, increase γH2AX level, and double-stranded DNA breaks in vitro. Meanwhile, overexpressed KLC2 significantly increased the proportion of the S phase in lung cancer cells. KLC2 knockdown could activate P53 pathway, and ultimately promoting radiosensitivity. The mRNA of KLC2 was observed to bind with Hu-antigen R (HuR). The mRNA and protein expression of KLC2 in lung cancer cells was significantly reduced when combined with siRNA-HuR. Interestingly, KLC2 overexpression significantly increased the expression of HuR in lung cancer cells. Taken together, these results indicated that HuR-KLC2 forms a positive feedback loop, which decreases the phosphorylation of p53 and thereby weaken the radiosensitivity of lung cancer cells. Our findings highlight the potential prognosis and therapeutic target value of KLC2 in lung cancer patients treated with radiotherapy.

The novel polyfluoroalkyl benzenesulfonate OBS exposure induces cell cycle arrest and senescence of rat pituitary cell GH3 via the p53/p21/RB pathway

Sodium p-perfluorous nonenoxybenzene sulfonate (OBS), an economical alternative to perfluorooctane sulfonate (PFOS) in multiple industrial fields, is widely detected in the environment. The toxicity of OBS has received increasing attention. Pituitary cells are components of the endocrine system and act as vital regulators of homeostatic endocrine balance. However, the effects of OBS on pituitary cells remain unknown. The present study explores the effects of OBS (0.5, 5, and 50 μM) on GH3 rat pituitary cells after treatment for 24, 48, and 72 h. We found that OBS significantly inhibited cell proliferation in GH3 cells with remarkable senescent phenotypes, including enhanced SA-β-gal activity and expression of senescence-associated secretory phenotype (SASP)-related genes, cell cycle arrest, and upregulation of the senescence-related proteins γ-H2A.X and Bcl-2. OBS caused significant cell cycle arrest of GH3 cells at the G1-phase and concomitantly downregulated the expression of some key proteins for the G1/S transition, including cyclin D1 and cyclin E1. Consistently, the phosphorylation of retinoblastoma (RB), which plays a central role in regulating the cell cycle, was prominently reduced after OBS exposure. Furthermore, OBS notably activated the p53-p21 signalling pathway in GH3 cells, as evidenced by increased p53 and p21 expressions, enhanced p53 phosphorylation, and augmented p53 nuclear import. To our knowledge, this study is the first to reveal that OBS triggers senescence in pituitary cells via the p53-p21-RB signalling pathway. Our study demonstrates a novel toxic effect of OBS in vitro, and provides new perspectives for understanding the potential toxicity of OBS.

VprBP/DCAF1 regulates p53 function and stability through site-specific phosphorylation

VprBP (also known as DCAF1) is a recently identified kinase that is overexpressed in cancer cells and serves as a major determinant for epigenetic gene silencing and tumorigenesis. The role of VprBP in driving target gene inactivation has been largely attributed to its ability to mediate histone H2A phosphorylation. However, whether VprBP also phosphorylates non-histone proteins and whether these phosphorylation events drive oncogenic signaling pathways have not been explored. Here we report that serine 367 phosphorylation (S367p) of p53 by VprBP is a key player in attenuating p53 transcriptional and growth suppressive activities. VprBP catalyzes p53S367p through a direct interaction with the C-terminal domain of p53. Mechanistically, VprBP-mediated S367p inhibits p53 function in the wake of promoting p53 proteasomal degradation, because blocking p53S367p increases p53 protein levels, thereby enhancing p53 transactivation. Furthermore, abrogation of VprBP-p53 interaction by p53 acetylation is critical for preventing p53S367p and potentiating p53 function in response to DNA damage. Together, our findings establish VprBP-mediated S367p as a negative regulator of p53 function and identify a previously uncharacterized mechanism by which S367p modulates p53 stability.

Melatonin inhibits senescence-associated melanin pigmentation through the p53-TYR pathway in human primary melanocytes and the skin of C57BL/6 J mice after UVB irradiation

UVB exposure accelerates skin aging and pigmentation. Melatonin effectively regulates tyrosinase (TYR) activity and aging. The purpose of this study was to determine the association between premature senescence and pigmentation, and the mechanism of melanin synthesis effected by melatonin. Primary melanocytes were extracted and identified from the male foreskin. To inhibit TYR expression, primary melanocytes were transduced with the lentivirus pLKD-CMV-EGFP-2A-Puro-U6-TYR. The wild-type TYR(+/+) and TYR(–/–) or TYR(+/–) knockout C57BL/6 J mice were used to determine the role of TYR on melanin synthesis in vivo. Results showed that UVB-induced melanin synthesis is dependent on TYR in primary melanocytes and mice. Furthermore, in primary melanocytes pretreated with Nutlin-3 or PFT-α to up or downregulate p53, results showed that premature senescence and melanin synthesis increased in primary melanocytes after UVB irradiation at 80 mJ/cm2, and further increased after being treated with Nutlin-3, while significantly decreased with PFT-α. In addition, melatonin inhibited UVB-induced premature senescence associated with inactivation of p53 and phosphorylation of p53 on Ser15 (ser-15), a decrease of melanin synthesis accompanied by reduced TYR expression. Moreover, skin erythema and pigmentation induced by UVB were reduced in the dorsal and ear skin of mice topically pretreated with 2.5% melatonin. These indicate that melatonin inhibits UVB-induced senescence-associated pigmentation via the p53-TYR pathway in primary melanocytes and prevents pigmentation obviously in the dorsal and ear skin of C57BL/6 J mice after UVB irradiation.Key messagesP53 links UVB irradiation-induced senescence and senescence-associated pigmentation and regulates TYR in primary melanocytes after UVB irradiation.Melatonin inhibits senescence-associated pigmentation through the p53-TYR pathway in primary melanocytes. Melatonin prevents skin erythema and melanin pigmentation induced by UVB irradiation in the dorsal and ear skin of C57BL/6J mice.

Abstract 1554: IpY.20, a first-in-class inhibitor of p27Kip1, inhibits CDK4/6 and CDK2 to kill tumor cells

Abstract The drug resistance seen in the presence of CDK4/6 inhibitors, such as palbociclib, can be attributed to compensatory activation of tumor cell proliferation by CDK2. While combination therapy of CDK4/6i with Estrogen modulators significantly extends Progression Free Survival (PFS) in patients with metastatic HR+ breast cancer, their tumors invariably develop resistance, which results in only marginal improvement in their Overall Survival (OS). To address this unmet medical need, both CDK4/6 and CDK2 must be inhibited simultaneously. p27Kip1, the natural inhibitor of CDK4/6 and CDK2, is responsible for turning them ON and OFF during periods of proliferation and quiescence, respectively. The transition from ON to OFF state is mediated by a specific modification to p27 itself, by the tyrosine kinase BRK (breast tumor related kinase). A naturally-occurring ALTernatively spliced form of BRK, named ALT-BRK or ALT, can bind to p27, block BRK's association and prevent BRK's phosphorylation of p27. The binding of ALT to BRK locks both CDK4/6 and CDK2 into the OFF conformation and simultaneously inhibits activities of both kinases. We engineered IpY.20, a smaller and more stable variant of ALT that is formulated within a liposome, to increase its serum residence time and facilitate delivery into tumor cells. IpY.20 inhibits cell proliferation of HR+ and triple negative (TN) breast cancer, ovarian, uterine and NSCLC, preferentially over non-tumor cells, supporting its clinical utility. Unlike CDK4/6 and CDK2i which only cause cytostasis, IpY.20 arrests proliferation of CDK4/6i-resistant and endocrine-resistant BC cells and induces cell death, demonstrating that p27 inhibition is a viable therapeutic approach to combat drug resistance due to resurgence of tumor cell growth due to escape from quiescence. In vivo, IpY.20 reduces tumor volume and increases OS in cell line-derived xenografts (CDX). IpY.20 has several important differentiating features when compared to CDK4/6 or CDK2 inhibitors: targeting p27 is more selective with fewer off-target effects than the ATP-competitive small molecule CDK4/6i currently in use; IpY.20 induces tumor cell death as opposed to cytostasis; the specific form of tumor cell death that is induced upon IpY.20 treatment, i.e., necroptosis, stimulates the innate and adaptive immune system. As the bulk of drug resistance seen with CDK or RAS/MAPK pathway targeting drugs is a result of compensatory CDK2 activity, IpY.20 will be a valuable member of the clinical arsenal for patients with drug resistant tumors. Currently, a robust clinical manufacturing process has been completed for IpY.20 formulation; along with a comprehensive characterization of nonclinical pharmacology, PK/PD, and tolerability, in preparation for the initiation of an IND-enabling program. Citation Format: Grace Chen, Lena Stafford, Anusha Aditya, Krishna Allamneni, Natalia Zisman, Hassan Rashidzadeh, Stacy W. Blain. IpY.20, a first-in-class inhibitor of p27Kip1, inhibits CDK4/6 and CDK2 to kill tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1554.

Abstract 2784: JW-1-283 inhibits melanoma tumor growth via the stabilization of p53 pathway

Abstract Melanoma is the most lethal skin cancer, with the increasing incidence in the past decade. Despite the significant advancements in immune therapy and targeted therapy, the overall prognosis for metastatic melanoma remains unsatisfactory. TP53 is a tumor suppressor, but its wildtype is inactivated in about 90% of melanoma. Since activated p53 can promote cancer cell apoptosis and thus effectively inhibit tumor growth and tumor metastasis, reactivation of p53 is considered a promising strategy for cancer therapy. Herein, we report a recently developed small molecule compound, JW-1-283, targeting the interactions between MDM2 E3 ubiquitin-protein ligase and p53 protein. In vitro, JW-1-283 showed potent cytotoxicity against A375 melanoma cell line (IC50 2.2±0.3 μM) which has wildtype p53, but its potency is substantially reduced in two other melanoma cell lines with either p53 mutation (M14) or p53 null (RPMI-7951) status. Treatment with JW-1-283 significantly dose-dependently reduced the stemness in A375 melanoma cells, as indicated by low numbers of colony formation, impaired tumor sphere formation abilities, and inhibition of cancer cell migration. Mechanistically, JW-1-283 disrupts the p53-MDM2 interaction, prevents p53 and MDM2 degradation in the existence of cycloheximide, and induces p38 and p53 phosphorylation in the apoptotic pathway. Genetic knockdown of p38 by siRNA further confirmed that the stabilized p53 could be subsequently phosphorylated by p38, leading to induced apoptosis as well as S phase cell cycle arrest. In vivo, when A375 tumors grown in NSG mice were treated with 7.5 mg/kg of JW-1-283 intraperitoneally every other day for a consecutive of 16 days, significant inhibition of tumor growth was observed, along with decreased tumor cell proliferation, tumor angiogenesis, and enhanced tumor cell apoptosis. In summary, this work provides the initial proof-of-concept of developing the JW-1-283 scaffold as a potential strategy for advanced melanoma, which inhibits tumor growth by activating the p53 signaling pathway. Citation Format: Yang Xie, Kelli Hartman, Zhongzhi Wu, Wei Li. JW-1-283 inhibits melanoma tumor growth via the stabilization of p53 pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2784.

Abstract 1632: MDM2 inhibitor alrizomadlin (APG-115) promotes antitumor activity of mitogen-activated protein kinase (MAPK) inhibitors in uveal melanoma

Abstract Uveal melanoma (UM) is the commonest primary intraocular malignancy, yet its molecular pathogenesis is poorly understood. Most UM cases have activating mutations in genes encoding G protein subunits alpha Q or 11, leading to activation of downstream effectors. These include protein kinase C, mitogen-activated protein kinases (MAPK1/3; also termed extracellular signal-regulated kinase 2/1 [ERK2⁄ERK1], respectively), and yes-associated protein, suggesting a rationale for therapeutically targeting these related pathways. Genetic analyses show that TP53 is infrequently mutated in UM, but associated pathways may be functionally inactivated. Ubiquitination-mediated degradation of p53 activates MAPK signaling, such that, active p53 may promote suppression of MAPK signaling. Alrizomadlin (APG-115) is a small molecule targeting p53/MDM2 that is in clinical development for solid and hematologic cancers. This study evaluated the antitumor effect of alrizomadlin, alone or combined with other targeted therapies, in preclinical models of UM. In cell-based antiproliferation assays of many UM cell lines, alrizomadlin exerted moderate growth inhibitory effects (IC50 = 0.151-1.857μM). When combined with inhibitors against mitogen-activated protein kinase kinase (MEK) [trametinib and selumetinib], mammalian target of rapamycin (mTOR) [rapamycin], focal adhesion kinase (FAK) [defactinib and Ascentage’s APG-2449, which targets FAK/ALK/ROS1], and MEK/rapidly accelerated fibrosarcoma [RAF] [VS-6766], alrizomadlin demonstrated enhanced antiproliferative activity. In a UM xenograft model (MP41), alrizomadlin showed dose-dependent antitumor activity when administered at 25, 50, and 100 mg/kg, with tumor growth inhibition (TGI) rates of 24.8%, 38%, and 39.1%, respectively. In this model, synergistic antitumor effects were observed when alrizomadlin was combined with selumetinib or APG-2449, with TGI rates of 65.4% and 47.5%, respectively. Mechanistically, alrizomadlin likely increased p53, MDM2, and downstream p21 in MP41 cells. Importantly, alrizomadlin inhibited ERK phosphorylation and enhanced selumetinib inhibition in a dose- and time-dependent manner. Prior studies indicated that FAK can suppress p53 by enhancing MDM2-dependent p53 ubiquitination. In this study, APG-2449 showed dose-dependent inhibition of FAK phosphorylation and upregulation of p53. Alrizomadlin also inhibited phosphorylation of FAK and downstream ERK. Therefore, the combination of alrizomadlin and APG-2449 was synergistic. Combining MEK and FAK inhibitors also markedly augmented caspase-3 and poly (ADP-ribose) polymerase 1 (PARP-1) cleavage (apoptosis hallmarks) and enhanced observed antitumor effects. In conclusion, our results demonstrate the potential utility of combining alrizomadlin with MAPK pathway inhibitors to treat patients with UM. Citation Format: Guangfeng Wang, Eric Liang, Ping Min, Dajun Yang, Yifan Zhai. MDM2 inhibitor alrizomadlin (APG-115) promotes antitumor activity of mitogen-activated protein kinase (MAPK) inhibitors in uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1632.

Abstract 458: Targeting p27Kip1 to inhibit CDK4/6/2 as a novel therapeutic strategy to treat pancreatic ductal adenocarcinoma

Abstract CDK4/6 inhibitors are powerful tools used clinically to treat metastatic, HR+ BC patients. While combined CDK4/6i treatment in combination with Estrogen modulation significantly extends Progression Free Survival (PFS) in metastatic HR+ breast cancer patients, those treated invariably develop aquired resistance, which results in only marginal improvement in Overall Survival (OS). The drug resistance seen in the presence of CDK4/6i, such as palbociclib, has been shown to be attributed to activation and compensation by CDK2. Pancreatic ductal adenocarcinoma (PDAC) is characterized by aberrant activation of the CyclinD-CDK4/6 signaling pathway and inactivation of the CDKN2A locus encoding the CDK4/6 inhibitor, p16INK4A, and was initially thought to be a good candidate for CDK4/6i use. Surprisingly, CDK4/6i do not show response in most PDAC lines and have not been used clinically in this tumor type. We hypothesized that PDAC may be intrinsically resistant to CDK4/6 inhibition due to activation of CDK2 and as such inhibition of both CDK4/6 and CDK2 may be required. We have developed a novel strategy to kill drug-resistant tumor cells using a therapeutic liposomal:peptide formulation, IpY.1, to inhibit the tyrosine phosphorylation of p27Kip1(CDKN1B), which in turn inhibits both CDK4/6 and CDK2. IpY.1 is specific for p27, and this reduces toxicity relative to that seen with the small molecule ATP-competitive CDK4/6i or CDK2i. IpY.1 blocks DNA replication in HR+ breast cancer (BC) cells and mouse models, but unlike CDK4/6i or CDK2i treatment, it induces ROS-dependent necroptosis to kill the tumor cells. Palbociclib did not have a significative effect on ATP levels or proliferation of PDAC cell lines. In contrast, we found that IpY.1 was able to block proliferation and reduce viability in palbociclib-resistant PDAC lines, suggesting that dual CDK4/6 and CDK2 inhibition via p27 targeting was required. Some PDAC lines were “super sensitive” and responded with IC50 values ~10X less than seen with the ER+ BC lines, and we are currently screening for biomarkers of response. CDK2 activity was reduced in IpY.1 treated lines and markers of necroptosis were detected. Immunoblot analysis showed decreased pY88 phosphorylation of p27 (the target of IpY) demonstrating target engagement. We have demonstrated that unlike palbociclib, IpY.1 blocks proliferation and viability in all PDAC lines tested, suggesting that targeting p27 may be a viable approach to treat PDAC. Citation Format: Carolina Guido, Susan RS Gottesman, Stacy W. Blain. Targeting p27Kip1 to inhibit CDK4/6/2 as a novel therapeutic strategy to treat pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 458.

Abstract 1429: C-terminal phosphorylation of p27 promotes cell cycle dependent changes through transcriptional coregulation of cJun and STAT3

Abstract p27 is a cyclin dependent kinase (CDK) inhibitor that binds cyclin-CDKs to arrest cell cycle. p27 undergoes PI3K/AKT-mediated C-terminal phosphorylation that alters its protein-protein interactions and function. Our prior work showed C-terminally phosphorylated p27 (p27pT157pT198) promotes its association with cJun. Genomic profiling showed p27 is co-recruited with cJun to over half of cJun chromatin binding sites to either activate or repress target genes. p27/cJun activated gene targets include TGFB2, and are associated with EMT, and programs that upregulate stem cells and alter cell adhesion and migration. Here, we pursued the hypothesis that cyclic changes in C-terminally phosphorylated p27 abundance govern p27-regulated transcriptional activity across the cell cycle, switching p27 from a corepressor of target genes in G0, to a transactivator upon PI3K/AKT activation in mid-G1. In synchronized NIH3T3 cells, we made the novel observation that p27pT197 phosphorylation is periodic, peaking in mid G1 after peak in Akt activation. Furthermore, phospho-activated cJunpS63, and phosphorylated STAT3 (STAT3pY705) levels are periodic across the cell cycle with maximal presence in mid-G1. cJun is minimally expressed in quiescence, and is strongly upregulated in early G1. While total cJun expression level remains constant at all time points after exiting G0, cJunpS63 rises sharply in G1 and then declines with cell cycle progression. Similarly, while STAT3 levels rise modestly on G0 exit, phosphorylated STAT3pY705 levels show a similar pattern to that of cJun pS63, minimal in quiescence, then increasing in mid G1. Moreover, both STATpY705 and cJunpS63 co-precipitation with total p27 and p27pT197 peak in early G1. Since complex formation is period, the action of p27/cJun/STAT3 on transcriptional regulation might also be periodic. Myc is upregulated early in G1 and is essential for G1 progression. ChIP-PCR indicate recruitment of p27 and H3K27 acetylation at MYC increased after exit from quiescence. RNA-seq of NIH3T3 cells across the cell cycle showed significant change of total transcript profiles very early after G0 exit and are distinct at each time point reflecting different phases of cell cycle. Genes governing ribosome biogenesis and protein synthetic machinery are rapidly upregulated in early G1. DNA replication/repair genes are rapidly downregulated in early G1 but rise with S/G2-M. Ongoing work investigates global target gene selection by p27/STAT3/cJun and their expression across the cell cycle, and will be presented. The potential role of p27-regulated gene expression in the coordinated progression of cells across the cell cycle has not been explored to date and might prove to be critical in cell cycle progression. Citation Format: Amir Bagheri, Seyedeh Fatemeh Razavipour, Madison Sharp, Joyce Slingerland. C-terminal phosphorylation of p27 promotes cell cycle dependent changes through transcriptional coregulation of cJun and STAT3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1429.

Abstract 306: Alternatively activated macrophages induce bystander effects via arginase 1

Abstract Colorectal cancer is a leading cause of cancer mortality and arises from genetic and epigenetic changes in colon epithelial cells. One of the mechanisms driving colon epithelial cell transformation is the bystander effect (BSE). BSE primarily arises from polarized macrophages (MØs) in stromal tissue. Herein, we report that alternatively activated (M2) MØs, as generated by interleukin (IL)-4, induce double-strand DNA breaks in target cells through BSE. Unlike M1 MØs that are activated by intestinal microbiota, BSE and DNA damage due to M2-like MØs occur through a distinct mechanism that differs from M1 MØs and does not involve pro-inflammatory mediators (e.g., cyclooxygenase-2 or tumor necrosis factor-α). Murine MØs (RAW264.7 cells) treated with IL-4 were polarized to an M2-like phenotype and found to strongly express arginase 1 (ARG1), a prototypical enzyme that converts L-arginine to L-ornithine and urea. L-Ornithine is a precursor in polyamine synthesis with H2O2 being a key oxidizing byproduct of polyamine catabolism. In a dual-chamber system using Young Adult Mouse Colon (YAMC) cells as targets for BSE generated by M2-like MØs, inhibition of ARG1 by N-hydroxy-nor-L-arginine abolished DNA damage (as measured by γH2AX). Direct treatment of YAMC cells with L-ornithine resulted in similar levels of DNA damage as M2-like MØs. YAMC cells exposed to M2-like MØs had increased intracellular H2O2. MDL-72527, an inhibitor polyamine oxidase and polyamine catabolism, reduced intracellular H2O2 to background levels and decreased DNA damage. YAMC cells sustaining DNA damage due to BSE from M2-like MØs showed specific phosphorylation of p53(ser15) and NF-κB(ser536) with no evidence for active caspase-3. These data along with an increased expression of cyclinD1 in YAMC cells indicated that DNA damage caused by M2-like MØs occurred via polyamine catabolism and was not due to cellular toxicity or apoptosis. Pathway analysis of RNAseq data for YAMC cells exposed to M2-like MØs identified multiple activated pathways including DNA repair, ubiquitination, Nrf2-mediated stress responses, endoplasmic reticulum overload responses, and lipid biosynthesis. These results provide additional evidence for global cellular responses to BSE generated by M2-like MØs. In conclusion, our findings show that M2-like MØs can generate BSE via a novel mechanism involving ARG1. Induction of BSE by M2-like MØs results in acute DNA damage in colon epithelial cells and activates multiple signaling pathways in target epithelial cells. These data imply a role for alternatively activated M2-like MØs in the malignant transformation of epithelial cells. Citation Format: Ram B. Undi, Hunter L. Porter, Jonathan D. Wren, Naushad Ali, Mark M. Huycke. Alternatively activated macrophages induce bystander effects via arginase 1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 306.

Abstract 2535: Inhibition of oncogenic ASPP2kappa(k), a dominant-negative isoform of the tumorsuppressor ASPP2, results in reactivation of p53 in glioblastoma

Abstract GB is the most common, most aggressive primary brain tumor with a deleterious prognosis. Novel approaches to improve therapy are urgently needed. ASPP2 is a tumor suppressor, directly interacting with p53 to induce apoptosis. We recently identified a dominant-negative isoform of ASPP2, named ASPP2kappa(k), lacking the important p53 binding sites. We now show that ASPP2k is highly expressed and functionally active in high grade glioma and that silencing of ASPP2k reactivates induction of apoptosis via p53. Freshly resected glioma (n=76) were assessed for ASPP2k expression. Isogenic, p53wt cell models, stably suppressing or overexpressing ASPP2k were established in A-172 and U-251 cells and ex vivo from GB samples and the role of ASPP2k in induction of apoptosis, cellular proliferation, migration and invasion was assessed. A proteome array allowed to study pathways involved in ASPP2k signaling. An ASPP2k knock-down, NOD/SCID xenotransplant mouse model was established to study the role of ASPP2k in tumor engraftment and progression. Frequent overexpression of ASPP2k in glioma was confirmed in virtually all patients analyzed. Expression patterns varied widely (up to 40-fold compared to tumor-free tissue). Expression of ASPP2k thereby correlates with tumor biology - with highest levels found in aggressive grade IV GB. We further demonstrate that expression of ASPP2k results in impaired induction of apoptosis in all GB models - and knock down of ASPP2k renders cells more susceptible towards temozolamide (TMZ), γ-irradiation (avg. 25% TMZ, 40% irradiation) or combinational approaches compared to controls. Vice versa, overexpression of ASPP2κ further attenuates induction of apoptosis. Even more, ASPP2k directly affects other cellular functions in GB, rendering cells to a more aggressive phenotype with increased proliferation (approx. 25%), migration (approx. 65%) and invasion rates compared to the knock down strains. In line, silencing of ASPP2k resulted in strong upregulation of crucial p53 phosphorylation sites (S15/S46), as well as cell cycle regulators (p21, p27 and pRAD) in a proteome array, arguing for re-activation of crucial p53-mediated pathways in the ASPP2k silenced cells. Results were confirmed by western immunoblotting and FACS analyses. Ongoing xenotransplant mouse models confirm that silencing of ASPP2k in GB models attenuates tumor engraftment, development and progression in vivo. In summary, we here show that ASPP2k is highly expressed in GB and directly contributes to aggressiveness of the disease as well as resistance towards therapy. Silencing of ASPP2k thereby re-activates crucial p53 pathways, resulting in re-activation of p53-mediated apoptosis pathways as well as re-installation of cell cycle checkpoints. Future studies evaluating ASPP2k as a prognostic/predictive model and potential target for therapy are warranted. Citation Format: Marlon Hafner, Alessia Ruiba, Leonie Kampa, Vincent Kälin, Marian Neidert, Konrad Buchauer, Guido Henke, Ludwig Plasswilm, Marcus M. Schittenhelmn, Kerstin M. Kampa-Schittenhelm. Inhibition of oncogenic ASPP2kappa(k), a dominant-negative isoform of the tumorsuppressor ASPP2, results in reactivation of p53 in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2535.

Echinochrome A Prevents Diabetic Nephropathy by Inhibiting the PKC-Iota Pathway and Enhancing Renal Mitochondrial Function in db/db Mice.

Echinochrome A (EchA) is a natural bioproduct extracted from sea urchins, and is an active component of the clinical drug, Histochrome®. EchA has antioxidant, anti-inflammatory, and antimicrobial effects. However, its effects on diabetic nephropathy (DN) remain poorly understood. In the present study, seven-week-old diabetic and obese db/db mice were injected with Histochrome (0.3 mL/kg/day; EchA equivalent of 3 mg/kg/day) intraperitoneally for 12 weeks, while db/db control mice and wild-type (WT) mice received an equal amount of sterile 0.9% saline. EchA improved glucose tolerance and reduced blood urea nitrogen (BUN) and serum creatinine levels but did not affect body weight. In addition, EchA decreased renal malondialdehyde (MDA) and lipid hydroperoxide levels, and increased ATP production. Histologically, EchA treatment ameliorated renal fibrosis. Mechanistically, EchA suppressed oxidative stress and fibrosis by inhibiting protein kinase C-iota (PKCι)/p38 mitogen-activated protein kinase (MAPK), downregulating p53 and c-Jun phosphorylation, attenuating NADPH oxidase 4 (NOX4), and transforming growth factor-beta 1 (TGFβ1) signaling. Moreover, EchA enhanced AMPK phosphorylation and nuclear factor erythroid-2-related factor 2 (NRF2)/heme oxygenase 1 (HO-1) signaling, improving mitochondrial function and antioxidant activity. Collectively, these findings demonstrate that EchA prevents DN by inhibiting PKCι/p38 MAPK and upregulating the AMPKα/NRF2/HO-1 signaling pathways in db/db mice, and may provide a therapeutic option for DN.

ISG20L1 acts as a co-activator of DAPK1 in the activation of the p53-dependent cell death pathway.

Our previous studies have revealed that GADD45α is a liable proapoptotic protein, which undergoes MDM2-dependent constitutive ubiquitylation and degradation in resting cancer cells. Under chemotherapeutic agent (such as arsenite, 5-Fu and VP-16) exposure, DAPK1 functions as a novel p53 (also known as TP53) kinase, which induces phosphorylation of p53 at Ser15 and transactivates the p53 target Ets-1, to synergistically repress IKKβ-dependent MDM2 stability, and ultimately removes the inhibitory effect of MDM2 on GADD45α, resulting in GADD45α accumulation and cell apoptosis. In the current study, we show that there is a strong induction of ISG20L1 (also known as AEN) expression in several cancer cell lines under exposure of arsenite and other chemotherapeutic agents. Surprisingly, although originally identified as a transcriptional target of p53, ISG20L1 induction was not controlled by p53. Instead, ISG20L1 functioned as upstream activator of p53 by interacting with DAPK1, and plays an essential role in promoting DAPK1-p53 complex formation and the subsequent activation of Ets-1/IKKβ/MDM2/GADD45α cascade. Therefore, our findings have revealed novel function of ISG20L1 in mediating cancer cell apoptosis induced by chemotherapeutic agents via modulating activation of the DAPK1- and p53-dependent cell death pathway.

Antioxidants prevent particulate matter-induced senescence of lung fibroblasts

Particulate matter (PM) contributes to human diseases, particularly lung disease; however, the molecular mechanism of its action is yet to be determined. Herein, we found that prolonged PM exposure induced the cellular senescence of normal lung fibroblasts via a DNA damage-mediated response. This PM-induced senescence (PM-IS) was only observed in lung fibroblasts but not in A549 lung adenocarcinoma cells. Mechanistic analysis revealed that reactive oxygen species (ROS) activate the DNA damage response signaling axis, increasing p53 phosphorylation, ultimately leading to cellular senescence via an increase in p21 expression without affecting the p16-pRB pathway. A549 cells, instead, were resistant to PM-IS due to the PM-induced ROS production suppression. Water-soluble antioxidants, such as vitamin C and N-Acetyl Cysteine, were found to alleviate PM-IS by suppressing ROS production, implying that antioxidants are a promising therapeutic intervention for PM-mediated lung pathogenesis.

A network pharmacology-based approach to explore the active ingredients and molecular mechanism of Shen-Kui-Tong-Mai granules on a rat model with chronic heart failure.

This study aimed to comprehensively investigate the potential active components and therapeutic mechanisms of Shen-Kui-Tong-Mai granule (SKTMG) in the treatment of heart failure. Network pharmacology combined with ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS), molecular docking, and in vivo validation was performed to identify the active components and the potential targets for SKTMG to improve chronic heart failure (CHF). The network pharmacology identified 192 active compounds and 307 potential consensus targets for SKTMG. On the other hand, network analysis discovered 10 core target genes related to the MAPK signal pathway. These genes include AKT1, STAT3, MAPK1, P53, SRC, JUN, TNF, APP, MAPK8 and IL6. The molecular docking results revealed that the SKTMG components were luteolin, quercetin, astragaloside IV and kaempferol, which could bind AKT1, MAPK1, P53, JUN, TNF and MAPK8. Additionally, SKTMG inhibited phosphorylation of AKT, P38, P53 and c-JUN, and reduced TNF-α expression in CHF rats. The present results demonstrated that network pharmacology combined with UHPLC-MS/MS, molecular docking and in vivo validation can facilitate the identification of active components and the potential targets for SKTMG to improve CHF.

Asperolide A induces apoptosis and cell cycle arrest of human hepatoma cells with p53-Y220C mutant through p38 mediating phosphorylation of p53 (S33)

Asperolides A (AA), one of the new tetranorlabdane diterpenoids, is proved to inhibit the proliferation of lung cancer cells and bone metastasis of breast cancer cells. Herein, we report that AA induces apoptosis and cell cycle arrest of hepatoma cells. It intensely inhibits proliferation of Huh-7 cell, compared with HepG-2 and L02 cells. AA elevates the activity of mitogen-activated protein kinases (MAPKs), in which the activation of ERK and JNK improves cell survival. However, phosphorylation of p53 at S33 by p38 activation could be a principal factor in the AA-induced apoptosis and G2/M cell cycle arrest of Huh-7 cells. The S33 site of p53-Y220C mutant, as the specific activation site of p38, reactivates the wild-type function of mutant p53 protein, which leads to a higher sensitivity of Huh-7 cells to AA. These results provide new insights into the molecular mechanisms of AA as a developing mutant p53 rescue drug.

DAPL1 prevents epithelial–mesenchymal transition in the retinal pigment epithelium and experimental proliferative vitreoretinopathy

Epithelial–mesenchymal transition (EMT) of the retinal pigment epithelium (RPE) is a hallmark of the pathogenesis of proliferative vitreoretinopathy (PVR) that can lead to severe vision loss. Nevertheless, the precise regulatory mechanisms underlying the pathogenesis of PVR remain largely unknown. Here, we show that the expression of death-associated protein-like 1 (DAPL1) is downregulated in PVR membranes and that DAPL1 deficiency promotes EMT in RPE cells in mice. In fact, adeno-associated virus (AAV)-mediated DAPL1 overexpression in RPE cells of Dapl1-deficient mice inhibited EMT in physiological and retinal-detachment states. In a rabbit model of PVR, ARPE-19 cells overexpressing DAPL1 showed reduced ability to induce experimental PVR, and AAV-mediated DAPL1 delivery attenuated the severity of experimental PVR. Furthermore, a mechanistic study revealed that DAPL1 promotes P21 phosphorylation and its stabilization partially through NFκB (RelA) in RPE cells, whereas the knockdown of P21 led to neutralizing effects on DAPL1-dependent EMT inhibition and enhanced the severity of experimental PVR. These results suggest that DAPL1 acts as a novel suppressor of RPE-EMT and has an important role in antagonizing the pathogenesis of experimental PVR. Hence, this finding has implications for understanding the mechanism of and potential therapeutic applications for PVR.

In vitro genotoxicity potential investigation of 7 oxy-PAHs.

Air pollutants include many compounds among them oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs). As they are suspected to generate DNA damage and mutagenicity, an understanding of their mode of action could highlight a carcinogenic potential risk in exposed population. In this article, a prospective study on seven oxy-PAHs selected in terms of occurrence in the environment was conducted on mutagenicity, genotoxicity, and cytotoxicity potentials using in vitro assays including Ames test on five strains, kinetic analysis of cytotoxicity and apoptosis, phosphorylation of histone H2AX, and p53 induction assays on human lung cell line BEAS-2B. Ames test demonstrated that mutagenicity pattern depended on the oxy-PAH tested. Except for BAQ, all oxy-PAHs tested gave mutagenic effect, in the absence and/or in the presence of metabolic activation (S9 fraction). At 24 h of exposure, the majority of oxy-PAHs induced γ-H2AX in BEAS-2B cells and/or phosphorylation of p53 at serine 15 and cell death at highest tested concentrations. Although 9,10-AQ and B[b]FO were mutagenic in bacteria, they failed to induce any of the other genotoxicity biomarkers. In comparison with the benzo[a]pyrene, all oxy-PAHs were less potent in terms of genotoxic potential at the same concentration. These results highlighted the genotoxic and mutagenic potential of these oxy-PAHs and provide preliminary information concerning their possible mechanism of action for toxicity, contributing to a better evaluation of the real associated health risks for human and environment.

EGFR and p38MAPK Contribute to the Apoptotic Effect of the Recombinant Lectin from Tepary Bean (Phaseolus acutifolius) in Colon Cancer Cells.

Previous works showed that a Tepary bean lectin fraction (TBLF) induced apoptosis on colon cancer cells and inhibited early colonic tumorigenesis. One Tepary bean (TB) lectin was expressed in Pichia pastoris (rTBL-1), exhibiting similarities to one native lectin, where its molecular structure and in silico recognition of cancer-type N-glycoconjugates were confirmed. This work aimed to determine whether rTBL-1 retained its bioactive properties and if its apoptotic effect was related to EGFR pathways by studying its cytotoxic effect on colon cancer cells. Similar apoptotic effects of rTBL-1 with respect to TBLF were observed for cleaved PARP-1 and caspase 3, and cell cycle G0/G1 arrest and decreased S phase were observed for both treatments. Apoptosis induction on SW-480 cells was confirmed by testing HA2X, p53 phosphorylation, nuclear fragmentation, and apoptotic bodies. rTBL-1 increased EGFR phosphorylation but also its degradation by the lysosomal route. Phospho-p38 increased in a concentration- and time-dependent manner, matching apoptotic markers, and STAT1 showed activation after rTBL-1 treatment. The results show that part of the rTBL-1 mechanism of action is related to p38 MAPK signaling. Future work will focus further on the target molecules of this recombinant lectin against colon cancer.

Microtubule-associated proteins MAP7 and MAP7D1 promote DNA double-strand break repair in the G1 cell cycle phase

The DNA-damage response is a complex signaling network that guards genomic integrity. The microtubule cytoskeleton is involved in the repair of DNA double-strand breaks; however, little is known about which cytoskeleton-related proteins are involved in DNA repair and how. Using quantitative proteomics, we discovered that microtubule associated proteins MAP7 and MAP7D1 interact with several DNA repair proteins including DNA double-strand break repair proteins RAD50, BRCA1 and 53BP1. We observed that downregulation of MAP7 and MAP7D1 leads to increased phosphorylation of p53 after γ-irradiation. Moreover, we determined that the downregulation of MAP7D1 leads to a strong G1 arrest and that the downregulation of MAP7 and MAP7D1 in G1 arrested cells negatively affects DNA repair, recruitment of RAD50 to chromatin and localization of 53BP1 to the sites of damage. These findings describe for the first time a novel function of MAP7 and MAP7D1 in cell cycle regulation and repair of DNA double-strand breaks.