P53 Expressing Research Articles

Comparison of PD-L1, VISTA, LAG-3, and GAL-3 Expressions and Their Relationships to Mismatch Repair Protein and p53 Expression in 529 Cases of Endometrial Carcinoma.

The aim of this study is to evaluate the expressions of programmed death-ligand 1 (PD-L1), V-domain Ig suppressor of T-cell activation (VISTA), lymphocyte activation gene-3 (LAG-3), and galectin-3 (GAL-3), in mismatch repair-deficient (MMRd)/MMR-proficient and abnormal p53 expressing endometrial carcinomas and their relationship with clinical-histopathological features. Patients who underwent surgery for endometrial carcinoma between January 2008 and December 2018 were included in the study. Immunohistochemical analysis of MLH1, PMS2, MSH2, MSH6, p53, PD-L1, VISTA, LAG-3, and GAL-3 was performed on the tissue samples of microarray. A total of 529 patients were included. MMRd and p53-mutant tumors accounted for 31.5% and 11.5% of cases, respectively. PD-L1 and LAG-3 expressions in the MMRd and p53-mutant groups were higher than in the MMR-proficient group (P < 0.001). GAL-3 expression in the MMR-proficient group was statistically higher than in the MMRd and p53-mutant groups (P < 0.001). Mean age, grade, International Federation of Gynecology and Obstetrics stage, lymphovascular invasion, and lymph node metastasis were significantly higher in the p53-mutant group (P < 0.001). In the group with PD-L1 expression, nonendometrioid histologic type, tumor grade, and lymphovascular invasion were significantly higher (P < 0.001). Tumor grade, lymphovascular invasion, lymph node metastasis, and microcystic, elongated and fragmented pattern of invasion were significantly higher in the group with high VISTA expression (P < 0.05). Tumor grade was significantly higher in the group with LAG-3 expression (P < 0.001). Immunohistochemically determined subgroups and PD-L1, VISTA, LAG-3, and GAL-3 expression levels may be useful indicators of molecular features, and clinical outcomes also may have important implications for the development of targeted therapies in endometrial carcinoma.

Abstract 3022: GPR109A activation limits cell growth by inducing p53 and its targets in human colon cancer cells

Abstract Chemical carcinogens or xenobiotics present in the diet play a significant role in colon cancer (CC) formation. p53, a tumor suppressor and multi-functional protein, provides an important barrier against the initiation and maintenance of CC. p53 mutation or inactivation is observed in about 60% of CCs and about 90% of all cancers. Therefore, therapeutic avenues that will rejuvenate wild-type p53 or target mutant p53 should have a significant impact on the prevention and treatment of CC. GPR109A, a G-protein coupled receptor for the FDA-approved lipid-lowering drug Niacin (NA), bacterial metabolite Butyrate (BTR), and ketone body β-hydroxybutyrate (BHB), functions as a novel tumor suppressor in the colon by providing the molecular link between colonic bacteria and p53 function. GPR109A activation limits cAMP by inhibiting adenylate cyclase (AC), which induces RAS and its downstream growth-promoting signaling. cAMP also enhances the binding of MDM2 to p53 and induces the proteasomal degradation of p53. p53 is an NAD+-dependent molecule and deficiency of NA, the precursor of NAD+, impairs p53 function. We tested the role of GPR109A and its agonists (NA, BTR, and BHB) in normal immortalized colon epithelial cell lines (CCD33 and CCD81) and various CC lines such as HCT116-p53+/+, HCT116-p53−/−, DLD1 (p53 point mutation), VACO4A (p53 insertion mutation), and SW498 (p53 deletion) and measured cell proliferation, cell cycle, apoptosis, and analyzed the expression of GPR109A and p53 (total, phosphorylated, and acetylated), MDM2, p21, Bax, and Puma in the presence and absence of GPR109109 agonists. We found that GPR109A expression is significantly reduced in CC cell lines, especially in p53 mutant cell lines, when compared to normal immortalized colonic epithelial cell lines. Treatment of CC cell lines with GPR109A agonists-induced apoptosis in wild-type p53 expressing CC by activating p53 and its targets. However, some lesser extent in p53 mutant or p53 null cell lines. Lentiviral-mediated GPR109A expression in CC cell lines itself induced apoptosis in both wild-type and mutant p53 expressing CC cell lines and GPR109A agonists treatment induced further. Interestingly, GPR109A agonists BTR and BHB significantly increased apoptosis in p53 mutant and p53 null CC cell lines in a p53-independent manner. Overall, our results provide evidence that GPR109A activation can be used as an effective therapeutic strategy for the treatment of CC. Citation Format: Muthusamy Thangaraju, Nanditi N. Thangaraju, Lauren Gilstrap, Madison Chimenti, Tulshi Patel, Sathish Sivaprakasam, Nikhil Patel, Santhakumar Manicassamy, Puttur D. Prasad, Vadivel Ganapathy. GPR109A activation limits cell growth by inducing p53 and its targets in human colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3022.

Abstract 1706: Investigating the interaction between ETS family members and mutant p53

Abstract Cancer cases are on the rise globally requiring a deeper understanding of the disease and identification of novel therapeutic targets. Mutations in genes often result in tumor formation. TP53 encodes p53, a tumor suppressive transcription factor, and is mutated in nearly 50% of all cancer cases. Once mutated, p53 loses its tumor suppressive function while simultaneously gaining oncogenic function. p53 mutations disrupt direct p53 binding to DNA and promotes p53 binding to regulatory regions of oncogenes via protein-protein interactions with other transcription factors. One of these interacting partners is ETS2, a member of the ETS transcription factor family. ETS binding sites are present in 50% of all mutant p53 occupied regulatory elements. Other ETS family members have also been linked to mutant p53 but these interactions have either been deemed weak (ETS1) or have yet to be identified as direct (ERG). It is my aim to determine the scope of interaction between the ETS family and mutant p53 and to elucidate whether this interaction is required for the observed oncogenic phenotypes of ETS proteins and mutant p53.To determine which ETS proteins interact with mutant p53 purified ETS proteins were used for affinity pull-down of purified mutant p53. The entire ETS family interacted with mutant p53 to some degree, and several ETS proteins had stronger interactions than ETS2. Truncation studies were used to determine interaction domains. One interacting region was the conserved DNA binding domain, which may explain interaction across the entire family. ERG and ETS2, both strong mutant p53 interactors, had two interaction interfaces. I hypothesize that the second interaction interface defines strong interactors. To determine if interacting ETS are responsible for targeting mutant p53 to the genome, chromatin immunoprecipitation sequencing measured mutant p53 binding in the presence or absence of different ETS factors. These data indicate that each of the conditions resulted in different p53 binding patterns and that there is a requirement for ETS in mutant p53 binding. Lastly, I wanted to determine if there was a correlation between p53 mutation and expression of strong mutant p53 interactors in certain cancer types. Analysis of TCGA samples showed that in ovarian cancers with p53 mutations, ETS factors that are strong mutant p53 interactors were upregulated in a mutually exclusive pattern, while weak interactors tended to be downregulated, or unchanged. Similar trends were observed among some other cancer types. My studies have shown that several ETS proteins interact with mutant p53 in vitro, recruit p53 to the genome, and that this interaction pattern correlates with expression in mutant p53 driven cancers. Additionally, studies are ongoing to determine phenotypes related to this interaction. Ultimately, if ETS/mutant p53 interactions are deemed important for oncogenesis, these will be attractive targets for drug development. Citation Format: Stephanie Metcalf, Peter Hollenhorst. Investigating the interaction between ETS family members and mutant p53 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1706.

The p53-mediated cell cycle regulation is a potential mechanism for emodin-suppressing osteosarcoma cells

BackgroundAs the most common primary bone cancer, the therapy of osteosarcoma requires further study. An anthraquinone derivative, emodin, has been found to have anticancer potential. We proposed that emodin suppresses osteosarcoma by cell cycle regulation mediated by p53. MethodsThis study determined the effect of emodin on viability and apoptosis of 6 osteosarcoma cell lines (p53 null cells MG63, G292, and A-673; p53 mutated cells HOS and SK-PN-DW; p53 expressing cells U2OS and 2 osteoblast cell lines), then knockdown p53 in U2OS, and observed the impacts of emodin on p53, p21, cyclin proteins, and cell cycle. ResultsHigh dose emodin (40–160 μM) induced cell death and apoptosis of all the cell lines; medium dose emodin (20 μM) preferentially inhibited osteosarcoma cells; low dose emodin (1–10 μM) preferentially inhibited p53 expressing osteosarcoma cells. Emodin dose-dependently inhibited p53 and p21 in U2OS. Emodin at 10 μM decreased the expression of Cdk2, E2F, and Cdk1; and increased RB but had no effects on cyclin E and cyclin B. The knockdown of p53 almost eliminated all the impacts of 10 μM emodin on cell cycle proteins. ConclusionsEmodin suppresses U2OS by p53-mediated cell cycle regulation.

Patient-derived tumor organoids with p53 mutations, and not wild-type p53, are sensitive to synergistic combination PARP inhibitor treatment

Poly (ADP-ribose) polymerase inhibitors (PARPi) are used for patients with BRCA1/2 mutations, but patients with other mutations may benefit from PARPi treatment. Another mutation that is present in more cancers than BRCA1/2 is mutation to the TP53 gene. In 2D breast cancer cell lines, mutant p53 (mtp53) proteins tightly associate with replicating DNA and Poly (ADP-ribose) polymerase (PARP) protein. Combination drug treatment with the alkylating agent temozolomide and the PARPi talazoparib kills mtp53 expressing 2D grown breast cancer cell lines. We evaluated the sensitivity to the combination of temozolomide plus PARPi talazoparib treatment to breast and lung cancer patient-derived tumor organoids (PDTOs). The combination of the two drugs was synergistic for a cytotoxic response in PDTOs with mtp53 but not for PDTOs with wtp53. The combination of talazoparib and temozolomide induced more DNA double-strand breaks in mtp53 expressing organoids than in wild-type p53 expressing organoids as shown by increased γ-H2AX protein expression. Moreover, breast cancer tissue microarrays (TMAs) showed a positive correlation between stable p53 and high PARP1 expression in sub-groups of breast cancers, which may indicate sub-classes of breast cancers sensitive to PARPi therapy. These results suggest that mtp53 could be a biomarker to predict response to the combination of PARPi talazoparib-temozolomide treatment.

Prognostic and Therapeutic Implications of TP53 expression in Chronic Myelomonocytic Leukaemia: Results of a Multicentre Study

Thetumor suppressor TP53 is among the most mutated genes in cancer, including many hematological malignancies in which it usually imparts more aggressive, chemo-refractory, and poor prognosis diseases. TP53 mutations are, though, remarkably rare in chronic myelomonocytic leukemia (CMML): affecting 2.4% of cases in a recent study ( Gurney et al, Leukemia 2023). We found pathogenic TP53 mutations ( TP53MT) in only 1.1% of 640 genotyped UK CMML patients. As expected, TP53MT conferred worse leukemia-free (LFS: median 23 v 28 mo, p=0.031) and overall (OS: 26 v 29 mo, p=0.015) survival compared with TP53WTcases. The clinical relevance in CMML of TP53 expression, however, remains unexplored. To address this we studied 92 TP53WT CMML patients (discovery cohort) from the National Taiwan University Hospital (Taipei, Taiwan) with available RNA-seq on presentation bone marrow (BM), each with extensive longitudinal clinical, laboratory and outcome data. Validation cohorts comprised 33 RNA-sequenced patients from The Christie (Manchester, UK) and 24 from Hospital Morales Meseguer (Murcia, Spain). For all cohorts, RNA-seq data from healthy controls (HCs) were available. Patients in the discovery cohort were divided into low and high TP53 expression groups, determined by the maximally selected rank statistics. TP53low patients (n=15) displayed significantly lower expression than HCs, while TP53high (n=77) had expression levels comparable to HCs. Thus, although most CMML patients are TP53WT, a subset has down-regulated TP53 expression and potential for altered p53 (and downstream) functions. TP53low and TP53high patients did not differ in clinical features or concurrent genetic alterations, although in all cohorts TP53low patients showed a trend towards poorer response to hypomethylating agents (HMA). TP53 low patients displayed significantly worse outcomes than TP53high patients (LFS 7 v 19 mo, p=0.002; OS 11 v 26 mo, p=0.001). Moreover, TP53 expression further stratified prognosis within different CMML-specific prognostic scoring system (CPSS)-molecular risk groups (Fig 1) . Time-dependent ROC analysis indicated TP53 expression as complementary to current risk stratification systems. In multivariable analysis, low TP53 expression remained prognostically detrimental. Survival analyses were consistent in both validation cohorts. GSEA and IPA analyses revealed that compared with HCs or TP53high, TP53low cells exhibited depleted expression of p53-dependent pathways, including MYC targets, E2F targets, G2/M checkpoints, and DNA repair. Interestingly, these are all among the most upregulated pathways in TP53MT (v TP53WT) samples across multiple cancers in TCGA data ( Donehower et al, Cell Rep 2019), indicating distinct roles for p53 in regulating these pathways in a TP53WT context, and that the driving biology of TP53low CMML is distinct from (and not functionally equivalent to) that of oncogenic TP53 mutations. TP53low patients demonstrated significantly enhanced TNF-alpha and inflammatory response signals, highlighting other distinctive pathobiology of the TP53low subgroup. In line with our clinical observation, single-sample GSEA showed significant enrichment of HMA resistance signatures in TP53low patients, potentially contributing to the dismal prognosis in this group. We next investigated whether this could be exploited therapeutically. We treated primary CMML BM cells ex vivo with azacitidine and NSC-207895, an MDMX inhibitor with p53 activating properties, observing clear and substantial synergy for combination therapy in samples from 10 of 11 patients. There was a trend towards inverse correlation between TP53 expression and empirical synergy scores (r=-0.35), suggesting potential for p53 activation to enhance HMA sensitivity in CMML, with broad efficacy, perhaps preferentially in adverse risk TP53low cases. In summary, we have identified and validated, across three cohorts from three countries, that a subgroup of CMML patients display aberrantly low TP53 expression, associated with HMA resistance and worse survival. Transcriptomic analyses revealed distinctive biology driving this novel TP53low subgroup (Fig 2), mandating further mechanistic study. Importantly we report consistent and substantial synergy for combining pharmacological p53 activation with HMA, presenting a novel targeted approach to improve HMA response for this unmet clinical need.

Augmented antitumor effects of erlotinib and cabozantinib on A549 non-small cell lung cancer: In vitro and in vivo studies

Non-small cell lung carcinoma is a challenging disease worldwide. This study aims to determine whether combining erlotinib, an epidermal growth factor receptor (EGFR) inhibitor, with cabozantinib, a mesenchymal-epithelial transition factor (c-Met) inhibitor, would have an augmented therapeutic benefit on A549 cells. The combination of erlotinib and cabozantinib (5 µM) inhibited A549 cell viability compared to each monotherapy at ≥ 10 µM as confirmed by the MTT assay. Combination therapy also has a more potent inhibition of cellular migration than monotherapy using the wound-healing assay. Furthermore, mRNA expression analyses for assessing apoptosis, metastasis, and cell cycle-related genes, the results showed that combination therapy significantly inhibits levels of BCL-2, MMP-9, VEGF, and TGF-β while inducing p53, p21, and BAX expression. In terms of oncogenic markers, western blotting analysis showed a significant reduction of BCl-2 expression and elevation in caspase3, p53, and p21 proteins as indicators of cell death via apoptosis. The antitumor in vivo effect of the combination therapy showed significant tumor inhibition compared to monotherapy. In conclusion, combination therapy could be a potential promising strategy to treat non-small cell lung carcinoma.

Differences and Similarities between Colorectal Cancer Cells and Colorectal Cancer Stem Cells: Molecular Insights and Implications.

Malignant tumors are formed by diverse groups of cancer cells. Cancer stem cells (CSCs) are a subpopulation of heterogeneous cells identified in tumors that have the ability to self-renew and differentiate. Colorectal cancer (CRC), the third most frequent malignant tumor, is progressively being supported by evidence suggesting that CSCs are crucial in cancer development. We aim to identify molecular differences between CRC cells and CRC CSCs, as well as the effects of those differences on cell behavior in terms of migration, EMT, pluripotency, morphology, cell cycle/control, and epigenetic characteristics. The HT-29 cell line (human colorectal adenocarcinoma) and HT-29 CSCs (HT-29 CD133+/CD44+ cells) were cultured for 72 h. The levels of E-cadherin, KLF4, p53, p21, p16, cyclin D2, HDAC9, and P300 protein expression were determined using immunohistochemistry staining. The migration of cells was assessed by employing the scratch assay technique. Additionally, the scanning electron microscopy method was used to examine the morphological features of the cells, and their peripheral/central elemental ratios were compared with the help of EDS. Furthermore, a Muse cell cycle kit was utilized to determine the cell cycle analysis. The HT-29 CSC group exhibited high levels of expression for E-cadherin, p53, p21, p16, cyclin D2, HDAC9, and P300, whereas KLF4 was found to be high in the HT-29. The two groups did not exhibit any statistically significant differences in the percentages of cell cycle phases. The identification of specific CSC characteristics will allow for earlier cancer detection and the development of more effective precision oncology options.

The NRF2-p97-NRF2 negative feedback loop

p97 is a ubiquitin-targeted ATP-dependent segregase that regulates proteostasis, in addition to a variety of other cellular functions. Previously, we demonstrated that p97 negatively regulates NRF2 by extracting ubiquitylated NRF2 from the KEAP1-CUL3-RBX1 E3 ubiquitin ligase complex, facilitating proteasomal destruction. In the current study, we identified p97 as an NRF2-target gene that contains a functional ARE, indicating the presence of an NRF2-p97-NRF2 negative feedback loop that maintains redox homeostasis. Using CRISPR/Cas9 genome editing, we generated endogenous p97 ARE-mutated BEAS-2B cell lines. These p97 ARE-mutated cell lines exhibit altered expression of p97 and NRF2, as well as a compromised response to NRF2 inducers. Importantly, we also found a positive correlation between NRF2 activation and p97 expression in human cancer patients. Finally, using chronic arsenic-transformed cell lines, we demonstrated a synergistic effect of NRF2 and p97 inhibition in killing cancer cells with high NRF2 and p97 expression. Our study suggests dual upregulation of NRF2 and p97 occurs in certain types of cancers, suggesting that inhibition of both NRF2 and p97 could be a promising treatment strategy for stratified cancer patients.

Epigenetic upregulation of spleen tyrosine kinase in cancer cells through p53-dependent downregulation of DNA methyltransferase

Syk is a tumor suppressor gene in some solid tumors. Currently, it remains unknown how Syk gene hypermethylation is controlled by DNA methyltransferase (DNMT) and p53. In colorectal cancer HCT116 cells, we found that protein and mRNA levels of Syk were much higher in WT than in p53−/− cells. Both p53 inhibitor PFT-α and p53 silencing can reduce the protein and mRNA expression of Syk in WT cells, while DNMT inhibitor 5-Aza-2′-dC can increase Syk expression in p53−/− cells. Interestingly, the DNMT expression in p53−/− HCT116 cells was higher than that in WT cells. PFT-α can not only enhance Syk gene methylation but also increase DNMT1 protein and mRNA levels in WT HCT116 cells. In metastatic lung cancer cell lines A549 and PC9, which express WT p53 and gain function of p53, respectively, PFT-α can also downregulate Syk mRNA and protein expression. However, the Syk methylation level was increased by PFT-α in A549 but not in PC9 cells. Likewise, 5-Aza-2′-dC transcriptionally increased Syk gene expression in A549 cells, but not in PC9 cells. In summary methylation of Syk promoter requires DNMT1, and p53 can upregulate Syk expression via downregulation of DNMT1 at the transcriptional level.

LncRNA STK4 antisense RNA 1 (STK4-AS1) promoted osteosarcoma by inhibiting p53 expression.

LncRNA STK4 antisense RNA 1 (STK4-AS1) has been identified as a potential biomarker associated with multiple cancers. We proposed that STK4-AS1 plays a role in the proliferation of osteosarcoma by regulating the cell cycle. We compared the expression of STK4-AS1, p53, and p21 in osteosarcoma vs normal samples in clinical tissues and cell lines. We determined the effect of overexpression and knockdown of STK4-AS1 in p53 expressing osteosarcoma cells U2OS, p53 muted osteosarcoma cells MG63, and osteoblast cells hFOB on p53 and p21 expression and the cell viability. For U2OS and MG63, the cell cycle was analyzed and the expression of cyclin proteins was determined. We overexpressed p53 or p21 in STK4-AS1 overexpressed cells to explore the association of STK4-AS1 and p53 in U2OS. The STK4-AS1 expression was higher and p53 and p21 expression were lower in osteosarcoma tissue and cells than in their non-cancer counterparts. The expression of STK4-AS1 was negatively correlated with the expression of p53 or p21. Knockdown of STK4-AS1 in U2OS decreased the cell viability, increased cells in the G0/G1 phase, decreased cells in the S and G2/M phase, decreased expression of cyclin A and B, increased p53 and p21, and had no effect on cyclin D and cyclin E, while overexpression of STK4-AS1 did the opposes. Overexpression of p53 or p21 recovered some changes caused by STK4-AS1 overexpression in U2OS. MG63 expressed no p53 and the expression of p21, cyclin A, and cyclin B, cell viability, and cell cycle were not affected by altered STK4-AS1 levels. In hFOB cells, the expression of p53 and p21 was decreased and the cell viability was increased when STK4-AS1 was overexpressed, but they were not affected when STK4-AS1 was knocked down. LncRNA STK4-AS1 promoted the cell cycle of osteosarcoma cells by inhibiting p53 expression.

TMIC-15. AGE-RELATED MARKERS FOR SENESCENCE INCREASE IN THE OLDER ADULT EXTRATUMORAL MOUSE BRAIN DUE TO GLIOBLASTOMA

Abstract OBJECTIVE The median age of onset for glioblastoma (GBM; IDHwt) is 68-70 years. Age is a strong prognostic factor for GBM patient outcomes such that overall survival in older adults is less than their younger counterparts – even after adjustment for MGMT promoter methylation status. Aging is associated with increased levels of senescence in the brain. Several age-related neurological disorders have been shown to improve with senolytic treatments. Here, we explored the effects and therapeutic neutralization of syngeneic brain tumors on increasing senescence levels in the extratumoral brain (ie. outside of the brain tumor) of young and old mice. METHODS General RNA-sequencing, as well as single-cell (sc) RNA-sequencing was performed on extratumoral tissue from young (8-12 weeks) and older adult (80-90 weeks) C57BL/6 mice with or without GL261 and key markers were validated with RT-PCR. The combined effects of the senolytics, dasatinib and quercetin, with radiation, anti-PD-1 mAb, and IDO enzyme inhibitor treatment, was also investigated. RESULTS General- and sc-RNA sequencing revealed a distinct gene expression profile in the extratumoral brain of older mice with syngeneic GL261 as compared to all other groups. RT-PCR results confirmed that the brain tumor increased gene expression for senescence levels, p53, and NFkB signaling in the older adult extratumoral brain. Expression of the senescence marker p16INK4A was primarily localized to oligodendrocyte progenitor cells in the older adult brain. The combinatorial treatment of senolytics with RT, anti-PD-1 mAb, and IDO enzyme inhibitor led to a synergistic survival benefit in older adult mice with GL261 as compared to the treatment with senolytics, or immunotherapy, alone. CONCLUSIONS The data suggest that the extratumoral brain may be responsible in-part for the poorer outcomes of older adults with GBM and that treatment approaches that target senescent cells may provide clinical benefit.

Hemophagocytic Lymphohistiocytosis Associated with Diffuse Large B-Cell Lymphoma: A Case Report

Abstract Introduction/Objective Secondary hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome of immune hyperactivation and dysregulation that has been observed in some malignancies. In HLH, natural killer (NK) cells and cytotoxic T-cells fail to control activated macrophages, leading to cytokine-mediated tissue injury and multiorgan failure. Persistent tumor antigenic stimulation and cytokine hypersecretion can trigger HLH. Diffuse large B- cell lymphoma (DLBCL) is uncommonly associated with HLH. Methods/Case Report A 50-year-old woman presented with daily fevers and malaise of one-month duration. Imaging showed marked hepatosplenomegaly with no lymphadenopathy. Laboratory studies were significant for pancytopenia (WBC: 3.8 K/µL, Hb: 6.8 g/dL, Plt: 74 K/µL), hypertriglyceridemia (330 mg/dL), hyperferritinemia (1460 ng/mL), and elevated soluble interleukin-2 receptor (CD25) (42351.5 pg/mL). These findings, along with NK cell activity level (not obtained in this case), fever, splenomegaly and hemophagocytosis (bone marrow, spleen or lymph nodes) represent the eight diagnostic criteria by Histiocyte Society HLH-2004. A splenic biopsy showed a nodular proliferation of medium-to-large neoplastic lymphocytes with CD20, PAX5, BCL6, MUM1, BCL2, CD5 (dim) and CD10 (dim) expression and negative for cyclin D1 and p53. CD21 did not highlight any dendritic cell meshwork. The bone marrow biopsy provided similar results. Overall, these findings were consistent with a DLBCL, germinal center subtype. Rearrangements of MYC, BCL2 or BCL6 genes were not detected. Although hemophagocytosis was not noted in the bone marrow or spleen, the patient met six of the eight HLH-2004 criteria (minimum five are required). The H-score was 174 reflecting up to 70% probability of HLH. Treatment with daily intravenous dexamethasone 10 mg/m2 was initiated as a temporizing measure prior to R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone) chemotherapy which resulted in complete remission. Results (if a Case Study enter NA) NA Conclusion Physicians should be cognizant of secondary HLH as a rare complication of DLBCL. Prompt initiation of the appropriate therapy could result in rapid and complete resolution.

P53 Promotes Ferroptosis in Macrophages Treated with Fe3O4 Nanoparticles.

Fe3O4 nanoparticles are the most widely used magnetic nanoparticles in the biomedicine field. The biodistribution of most nanoparticles in vivo is determined by the capture of macrophages; however, the effects of nanoparticles on macrophages remain poorly understood. Here, we demonstrated that Fe3O4 nanoparticles could reduce macrophage viability after 48 h of treatment and induce a shift in macrophage polarization toward the M1 phenotype; RNA sequencing revealed the activation of the ferroptosis pathway and p53 upregulation compared to the control group. The expression in p53, xCT, glutathione peroxidase 4 (GPX4), and transferrin receptor (TFR) in macrophages was similar to that in erastin-induced ferroptosis in macrophages, and the ultrastructural morphology of mitochondria was consistent with that of erastin-treated cells. We used DCFH-DA to estimate the intracellular reactive oxygen species content in Fe3O4 nanoparticles treated with Ana-1 and JC-1 fluorescent probes to detect the mitochondrial membrane potential change; both showed to be time-dependent. Fer-1 inhibited the reduction of the glutathione/oxidized glutathione (GSH/GSSG) ratio and inhibited intracellular oxidative stress states; therefore, Fe3O4 nanoparticles induced ferroptosis in macrophages. Finally, we used pifithrin-α hydrobromide (PFT) as a p53 inhibitor to verify whether the high expression of p53 is involved in mediating this process. After PFT treatment, the live/dead cell rate, TFR, p53 expression, and GPX4 consumption were inhibited and mitigated the GSH/GSSG ratio reduction as well. This indicates that p53 may contribute to Fe3O4 nanoparticle-induced ferroptosis of macrophages. We provide a theoretical basis for the molecular mechanisms of ferroptosis in macrophages and the biotoxicity in vivo induced by Fe3O4 nanoparticles.

Antidiabetic effects of marine brown algae Spatoglossum asperum J. Ag. ethanolic extract on 3T3-L1 Pre-Adipocytes activity an in vitro approach

Marine algae have emerged as good alternative source of medical, pharmaceutical, and food industries, it’s used for anticancer, anti-inflammatory, antioxidant, antimicrobial, etc., In this study, anti-diabetic effect of brown seaweed Spatoglossum asperum was focused, We aimed systematically to examine the ethanolic extract effects of S. asperum (EESA) as an anti-diabetic agent using 3T3-L1 in vitro assay, focused through molecular works; protein extraction, DNA isolation, gene analysis, western blot, ROS measurement, senescence associated β-galactosidase and deoxy glucose uptakes. In results for p21/β-actin H2O2+EESA treated cells showed very good potential results showed (0.44), same combination reduced the toxic level (0.60) in p53/β-actin, awfully superior results showed (0.39) in p16/β-actin. In adiponectin (1.16), tnf-α (0.68), il6 (0.42), RFU (211.5), same combination in COMET assay observed that DNA damage (4.95), glucose uptake noticed (68.45%), mRNA expression in p53 showed (0.07), p21 (1.8), adiponectin (3.25), tnf-α obtained (1.95), il6 expression obtained (2), p16 expression (2). From the results concluded that the capability to supplement glucose absorption in 3T3-L1 pre-adipocytes and augment glucose consumption activity through the appearance of glucose transporter type 4 (GLUT 4).

Immunohisto Chemical Expression of IDH1, ATRX and P53 in Low Grade Diffuse Gliomas at Armed Forces Institute of Pathology, Rawalpindi

Objective: To determine the immune histochemical expression of IDH1, ATRX and p53 in low-grade diffuse gliomas.&#x0D; Study Design: Cross-sectional study.&#x0D; Place and Duration of Study: Department of Histopathology, Armed Forces Institute of Pathology, Rawalpindi Pakistan, from Jan to Jun 2020.&#x0D; Methodology: Forty-two cases of low-grade diffuse glioma were included in the study. The cases were stratified into diffuse astrocytoma and oligodendroglioma based on results of immunohistochemistry expression for IDH1, ATRX and p53.&#x0D; Results: The frontal lobe was the most commonly affected site 14 (33.3%). Based on the results of immunohistochemistry expression, 26 cases (61.9%) were diagnosed as an oligodendroglioma, while 16 cases (38.1%) were diagnosed as diffuse astrocytoma. All cases were assigned who grade II out of IV. In oligodendrogliomas, all cases (100%) were IDH1 mutants with the retained expression of ATRX and wild type expression of p53. Among the 16 cases of diffuse astrocytoma, IDH1 was mutant in 12 cases (75%), ATRX expression was lost in all cases, and p53 was mutant in 6 cases (37.5%).&#x0D; Conclusion: The study showed that low-grade diffuse gliomas could be stratified and sub-classified in accordance with the World Health Organization (WHO) classification of CNS tumours (2016) successfully by application of immunohistochemistry for IDH1, ATRX and p53.

Abstract 725: Biomarkers of circulating vitamin D status and expression of tumor molecular markers Ki67, p53, and COX2 among colorectal cancer cases in the Southern Community Cohort Study

Abstract Introduction: Epidemiologic studies and controlled trials provide evidence that higher 25-hydroxyvitaminD is associated with improved survival in colorectal cancer (CRC), although there are limited data in African Americans (AAs), who are at greater risk for CRC mortality and vitamin D deficiency. Herein, we report associations between vitamin D serum biomarkers collected at cohort entry up to 15 years prior to CRC onset and CRC tumor subtypes defined by high expression of tumor markers (Ki67, p53, and COX2) that represent cellular processes (proliferation, apoptosis, and inflammation) affected by vitamin D. Importantly, higher expression of these markers reflects greater risk for metastasis and CRC-specific mortality. Methods: Data arise from the Southern Community Cohort Study, a cohort from the southeastern United States where the majority of participants are AA. Incident CRC cases with tumor sample were analyzed (n=104). Expression of cellular Ki67, p53, and COX2 (% positivity) were measured and categorized by literature-defined high vs. low cut-points (20%, 10%, and 20%, respectively). Logistic models adjusted for age, race, and sex were used to measure associations between vitamin D biomarkers: 25-hydroxyvitaminD, vitamin D binding protein (VDBP), and calculated free 25-hydroxyvitaminD (vitamin D not bound by VDBP) and the odds of a more aggressive tumor subtype, defined by higher cellular expression. Associations were assessed in the full sample and the subsample of AAs (n=70). Results: There were 52 tumors with high Ki67 expression, 37 with high p53, and 50 with high COX2. Fifteen tumors had high expression of all three markers. Cases were 55.8±0.4 (mean±SE) years at enrollment (range 40-79). The median 25-hydroxyvitaminD was lower in cases with high p53 expressing tumors compared to low expression (13.5 vs. 18.0 ng/mL, respectively, p = .03). Median values for other biomarkers were similar by tumor subtype (p &amp;gt .05). 25-hydroxyvitaminD was not associated with having a tumor with high Ki67 expression (odds ratio (OR) per 1 SD increase [95% CI] = 1.29 [0.85-1.95], p-trend = .23), high p53 expression (0.80 [0.52-1.23], p-trend = .30), or high COX2 expression (1.29 [0.83-2.00], p = .25). We observed null associations for VDBP, and free 25-hydroxyvitaminD with all tumor subtypes. Results were similar in AAs, although higher VDBP was associated with lower odds of high Ki67 tumors (0.45 [0.21-0.97], p-trend = .04 per 1 SD increase). Conclusion: In this sample of predominantly AA CRC cases, we observed no association between vitamin D biomarkers in stored serum and subsequent more aggressive CRC molecular subtypes. More work is needed to characterize the relationship between vitamin D and expression of CRC tumor markers, which may help clarify the protective role of maintaining adequate vitamin D levels in association with CRC. Citation Format: Thomas Lawler, Timothy Su, Qiuyin Cai, Mark Steinwandel, Wei Zheng, William J. Blot, Shaneda Warren Andersen. Biomarkers of circulating vitamin D status and expression of tumor molecular markers Ki67, p53, and COX2 among colorectal cancer cases in the Southern Community Cohort Study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 725.

MLH1, BRAF and p53 - searching for significant markers to predict evolution towards adenocarcinoma in colonic sessile serrated lesions.

Background and Aim: Colonic serrated lesions are premalignant lesions, using an alternative malignization pathway, including multiple genetic and epigenetic alterations, as: mismatch repair deficiency due to MutL homolog 1 (MLH1) promoter methylation, tumor protein p53 (TP53) mutations, activating mutations of v-Raf murine sarcoma viral oncogene homolog B (BRAF) and Kirsten rat sarcoma viral oncogene homolog (KRAS). Our study aims to evaluate MLH1, BRAF and p53 immunohistochemical (IHC) status in sessile serrated lesions (SSLs), with and without dysplasia. Materials and Methods: This is a retrospective case-control study including 20 SSLs with dysplasia and 20 SSLs without dysplasia (matching sex and age). IHC expression of MLH1, BRAF and p53 was evaluated as the percent of nuclear loss of MLH1, cytoplasmic positivity of BRAF and nuclear positivity of p53. Data concerning age, sex, localization of the lesion, dysplasia and IHC results were statistically processed using Microsoft Excel. Results: We had very polymorphous patterns of IHC expression for BRAF, MLH1 and p53, especially in the dysplastic group. Thus, two patients were BRAF+/MLH1-/p53+, three were BRAF+/MLH1-/p53-, one was BRAF+/MLH1+/p53- and six were BRAF+/MLH1+/p53+. Dysplastic lesions without BRAF mutation exhibited the following phenotype: one case BRAF-/MLH1-/p53+, four BRAF-/MLH1-/p53- and three BRAF-/MLH1+/p53+. In the control group (SSLs without dysplasia), there was a more homogenous distribution of cases: eight cases BRAF+/MLH1+/p53-, seven BRAF-/MLH1+/p53-, one BRAF-/MLH1-/p53+, two BRAF-/MLH1-/p53- and two BRAF-/MLH1+/p53+. Conclusions: There are more routes on the serrated pathway, with different mutations and time of acquisition of each genetic or epigenetic lesion with the same morphological result. These lesions should be stratified according to their risk to poor outcome and their need to further surveillance.

The p97-Nploc4 ATPase complex plays a role in muscle atrophy during cancer and amyotrophic lateral sclerosis.

BackgroundThe p97 complex participates in the degradation of muscle proteins during atrophy upon fasting or denervation interacting with different protein adaptors. We investigated whether and how it might also be involved in muscle wasting in cancer, where loss of appetite occurs, or amyotrophic lateral sclerosis (ALS), where motoneuron death causes muscle denervation and fatal paralysis.MethodsAs cancer cachexia models, we used mice bearing colon adenocarcinoma C26, human renal carcinoma RXF393, or Lewis lung carcinoma, with breast cancer 4T1‐injected mice as controls. As ALS models, we employed 129/SvHsd mice carrying the mutation G93A in human SOD1. The expression of p97 and its adaptors was analysed in their muscles by quantitative real‐time polymerase chain reaction (qPCR) and western blot. We electroporated plasmids into muscles or treated mice with disulfiram (DSF) to test the effects of inhibiting p97 and nuclear protein localization protein 4 (Nploc4), one of its adaptors, on atrophy.ResultsThe mRNA levels of p97 were induced by 1.5‐fold to 2‐fold in tibialis anterior (TA) of all the cachectic models but not in the non‐cachectic 4T1 tumour‐bearing mice (P ≤ 0.05). Similarly, p97 was high both in mRNA and protein in TA from 17‐week‐old SOD1G93A mice (P ≤ 0.01). Electroporation of a shRNA for murine p97 into mouse muscle reduced the fibre atrophy caused by C26 (P = 0.0003) or ALS (P ≤ 0.01). When we interrogated a microarray, we had previously generated for the expression of p97 adaptors, we found Derl1, Herpud1, Nploc4, Rnf31, and Hsp90ab1 induced in cachectic TA from C26‐mice (Fold change > 1.2, adjusted P ≤ 0.05). By qPCR, we validated their inductions in TA of cachectic and ALS models and selected Nploc4 as the one also induced at the protein level by 1.5‐fold (P ≤ 0.01). Electroporation of a CRISPR/Cas9 vector against Nploc4 into muscle reduced the fibre atrophy caused by C26 (P = 0.01) or ALS (P ≤ 0.0001). Because DSF uncouples p97 from Nploc4, we treated atrophying myotubes with DSF, and found accumulated mono and polyubiquitinated proteins and reduced degradation of long‐lived proteins by 35% (P ≤ 0.0001), including actin (P ≤ 0.05). DSF halves Nploc4 in the soluble muscle fraction (P ≤ 0.001) and given to C26‐bearing mice limited the body and muscle weight loss (P ≤ 0.05), with no effect on tumour growth.ConclusionsOverall, cancer cachexia and ALS seem to display similar mechanisms of muscle wasting at least at the catabolic level. The p97‐Nploc4 complex appears to have a crucial role in muscle atrophy during these disorders and disrupting this complex might serve as a novel drug strategy.

Pleural fluid metastasis of plasmacytoid urothelial carcinoma in comparison to micropapillary and conventional high-grade urothelial carcinoma: Cytologic and immuonohistochemical findings.

Plasmacytoid urothelial carcinoma (PUC) is a rare but clinically aggressive variant of high-grade urothelial carcinoma (HGUC). Cytological features include single plasmacytoid neoplastic cells with N:C ratio around 0.5, eccentric nuclei, nuclear hyperchromasia, irregular nuclear membrane, and vacuolated cytoplasm. Micropapillary urothelial carcinoma (MPUC) is another clinically aggressive variant of HGUC that shares some overlapping features of PUC. The diagnosis of these two aggressive variants in pleural effusions can be challenging due to features mimicking adenocarcinoma, unusual immunochemistry profile, and confusion with differential diagnoses, especially when pertinent clinical information is unavailable. We present report on one case each of pleural fluid metastasis of PUC and MPUC respectively, and compare the findings with that of a metastatic conventional HGUC originally thought to be metastatic adenocarcinoma. The diagnosis of PUC was confirmed with immunohistochemical studies showing expression for cytokeratin, GATA-3, uroplakin II, and CD138, diminished or loss of E-cadherin membranous expression, negative expression for p63, p53, Epicam-BerEP4, Epicam-MOC31, and p120. The diagnosis of MPUC was confirmed with immunostain profile similar to that of PUC except positive stain for E-cadherin, p120, and p53. The diagnosis of HGUC was confirmed with immunohistochemical studies showing expression for cytokeratin, GATA-3, uroplakin II, p63, Epicam-BerEP4 (focal weak), and Epicam-MOC31. Our cases of metastatic urothelial carcinoma showed features mimicking adenocarcinoma and others, especially the MPUC and HGUC were diagnosed without prior tissue diagnosis of urothelial carcinoma. This report emphasizes the cytohistological and immunohistochemical details of urothelial carcinoma involving effusion fluid and discusses potential pitfalls in diagnosis.