TMOD-28. MYC GENERATES AGGRESSIVE MEDULLOBLASTOMA BY HSP90 PATHWAY ACTIVATION AND ARF SILENCING

Medulloblastoma, the most common malignant pediatric brain tumor, often shows amplification or overexpression of the MYC transcription factor and arises in the presence of a functional p53 tumor suppressor protein. To elucidate the mechanism behind this inexplicable tumor development we generated an inducible, immunocompetent transgenic mouse model of MYC-expressing medulloblastoma. Aggressive tumors developed clonally in the presence of an unaltered p53 gene that molecularly resembled Group 3 medulloblastoma. Compared to MYCN-expressing medulloblastoma driven from the same promoter, we instead discovered pronounced and MIZ1-independent silencing of the ARF suppressor, which was also suppressed in MYC-amplified as compared to MYCN-amplified human medulloblastoma. While MYCN-driven tumor malignancy was more sensitive to ARF depletion, it dramatically increased metastatic spread of MYC-driven tumors. DNMT inhibition could restore ARF levels in MYC-expressing tumors but did not show any therapeutic advantage in tumors in vivo. Bioinformatics analysis further showed a strong correlation of the HSP90 pathway with MYC in human Group 3 MB and in the MYC-driven mouse model. The HSP90 inhibitor Onalespib showed significant selectivity for targeting MYC-driven as compared to MYCN-driven tumors. The drug promoted ARF restoration and increased the survival in our animal model which suggests that it could be potentially used in the treatment of MYC-driven ARF-silenced brain cancer patients.

The ORF45 protein of Kaposi Sarcoma-associated Herpesvirus (KSHV) is an inhibitor of p53 signaling during viral reactivation

Kaposi Sarcoma-associated herpesvirus (KSHV) is a carcinogenic double-stranded DNA virus and the etiological agent of Kaposi’s Sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman’s Disease (MCD). To prevent premature apoptosis and support its replication cycle, KSHV expresses a series of open reading frames (ORFs) that regulate signaling by the p53 tumor suppressor protein. Here we describe a novel viral inhibitor of p53 encoded by KSHV ORF45 and identify its mechanism of action. ORF45 binds to p53 and prevents its interactions with USP7, a p53 deubiquitinase. This results in decreased accumulation, localization of p53 to the cytoplasm, and diminished transcriptional activity. IMPORTANCE Unlike in other cancers, the tumor suppressor protein p53 is rarely mutated in Kaposi Sarcoma (KS). Rather, Kaposi Sarcoma-associated herpesvirus (KSHV) inactivates p53 through multiple viral proteins. One possible therapeutic approach to KS is the activation of p53, which would result in apoptosis and tumor regression. In this regard, it is important to understand all the mechanisms used by KSHV to modulate p53 signaling. This work describes a novel inhibitor of p53 signaling and a potential drug target, ORF45, and identifies the mechanisms of its action.

p53/p73 Protein Network in Colorectal Cancer and Other Human Malignancies

The p53 tumor suppressor protein is crucial for cell growth control and the maintenance of genomic stability. Later discovered, p63 and p73 share structural and functional similarity with p53. To understand the p53 pathways more profoundly, all family members should be considered. Each family member possesses two promoters and alternative translation initiation sites, and they undergo alternative splicing, generating multiple isoforms. The resulting isoforms have important roles in carcinogenesis, while their expression is dysregulated in several human tumors including colorectal carcinoma, which makes them potential targets in cancer treatment. Their activities arise, at least in part, from the ability to form tetramers that bind to specific DNA sequences and activate the transcription of target genes. In this review, we summarize the current understanding of the biological activities and regulation of the p53/p73 isoforms, highlighting their role in colorectal tumorigenesis. The analysis of the expression patterns of the p53/p73 isoforms in human cancers provides an important step in the improvement of cancer therapy. Furthermore, the interactions among the p53 family members which could modulate normal functions of the canonical p53 in tumor tissue are described. Lastly, we emphasize the importance of clinical studies to assess the significance of combining the deregulation of different members of the p53 family to define the outcome of the disease.

Functional interplay among thiol-based redox signaling, metabolism, and ferroptosis unveiled by a genetic variant of TP53

The p53 tumor suppressor protein is a transcription factor and master stress response mediator, and it is subject to reduction-oxidation (redox)-dependent regulation. The P47S variant of TP53, which exists primarily in African-descent populations, associates with an elevated abundance of low molecular weight (LMW) thiols, including glutathione (GSH) and coenzyme A (CoA). Here we show that S47 and P47 cells exhibit distinct metabolic profiles, controlled by their different redox states and expression of Activating Transcription Factor-4 (ATF4). We find that S47 cells exhibit decreased catabolic glycolysis but increased use of the pentose phosphate pathway (PPP), and an enhanced abundance of the antioxidant, NADPH. We identify ATF4 as differentially expressed in P47 and S47 cells and show that ATF4 can reverse the redox status and rescue metabolism of S47 cells, as well as increase sensitivity to ferroptosis. This adaptive metabolic switch is rapid, reversible, and accompanied by thiol-mediated changes in the structures and activities of key glycolytic signaling pathway proteins, including GAPDH and G6PD. The results presented here unveil the important functional interplay among pathways regulating thiol-redox status, metabolic adaptation, and cellular responses to oxidative stress.

Clinico-Histopathological and Immunohistochemical Study of Ruminant’s Cutaneous Papillomavirus in Iraq

The papilloma viruses are constituted of double-stranded DNA and are a more common lesion in ruminant’s skin in Iraq. The p53 tumor suppressor protein reveals an essential role in cell cycle control. This study intends to investigate the clinical, histopathological, and immunohistochemical features of cutaneous papilloma in ruminants in Iraq. Samples had been collected from a total of 10 animals (three cattle, three goats, and four sheep) with multiple papillomatosis lesions. The samples were processed for histopathological and immunohistochemical techniques. Clinically, the lesions appeared as multiple various sizes (0.5–11 cm), cauliflower exophytic masses on different parts of the animal’s body. The histopathological features of the epidermis granular layer revealed perinuclear vacuolation (koilocytosis) accompanied by various degrees of hypergranulosis, hyperkeratosis, acanthosis, orthokeratosis, and parakeratosis. Strong positive reaction for papillomavirus antigen was seen in both epidermal basal and granular layers in the immunohistochemical investigation (IHC). Moreover, all papilloma lesions revealed an intense positive p53 reaction in cytoplasmic and perinuclear of the basal and parabasal layers. In conclusion, this study described the papillomavirus lesions in bovine, ovine, and caprine animals, which were found in different parts areas of the affected animals. All lesions show similar histopathological features with minor variations. PV antigen and p53 protein expression showed positive results in immunohistochemistry that can be used as diagnostic markers for ruminant’s papilloma.

Green Tea Extract (Camellia sinensis) as a Potential Antitumoral Agent on Breast Cancer Cells (FS13-04-19)

Objectives We aimed to obtain a catechin-rich green tea extract (GTE) from Camellia sinensis and to evaluate its antitumor potential on human breast cancer. Green tea (GT) has been extensively studied for its antioxidant property, which is mainly attributed to catechins. These phenolic compounds regulate many cellular targets involved in cancer signaling pathways, including those mediated by p53 tumor suppressor protein. Methods Tea infusion was prepared on a ratio of 1 g:40 mL of boiling distilled water using a combination of temperature and time (70, 75, 80, 85, 90, 95 and 100 ºC/5, 10, 15 min). Infusions were evaluated by polyphenols content (Folin–Ciocalteu's reagent) and antioxidant potential (FRAP). Breast cancer cells (MDA-MB-231 and MCF-7) and non-tumoral cells (MCF-10A), were exposed to different concentrations of GTE (31.5 μg/mL to 1.0 mg/mL) during 24 h-48 h, and cell viability was assessed by Alamar Blue® and Trypan Blue assays. Viability was also acessed in the presence of a p53 protein inibitor, pifithrin-α. Immunocytochemistry and Western blotting analysis were performed to access the expression of p53. Results Our results demonstrated no influence of extraction condition in the total content of polyphenols and antioxidant potential of GT, and a new extract was obtained at 80 ºC/5 min, then freeze dried to be used in cell culture. After exposure for 24 h, GTE already promoted a cytotoxic effect, reducing viability of both breast cancer cell lines (IC50 MDA-MB-231 = 138,8 μg/mL; IC50 MCF-7 = 324,5 μg/mL) without cytotoxic effect on non-tumoral cells (IC50 MCF-10A = 10,097 μg/mL). In addition, GTE promotes an increase of p53 levels on treated MCF-7 cells, which express the wild-type form of the protein. Interestingly, an opposite phenomenon was evidenced in MDA-MB-231 cells, that express the mutated form of p53, in which protein levels seem to be lower in the presence of the extract. Conclusions Our data suggests that GTE has anticarcinogenic potential on breast cancer and it is possible that this capacity might be related with the modulation of p53 tumor suppressor protein. Funding Sources FAPERJ, FUNDAÇÃO DO CÂNCER, CAPES, CNPq.

Clinico-Histopathological and Immunohistochemistry Study of Ruminant’s Cutaneous Papillomavirus in Al-Muthanna Veterinary Hospital/ Iraq

Background: Papillomaviruses (PVs) are double-stranded DNA viruses and are more common in skin of ruminants in Iraq. A P53 (tumor suppressor protein) reveals an essential role in cell cycle control. This study aimed to describe the clinical, histopathological and immunohistochemical aspects of naturally occurring cutaneous ruminant’s papillomatosis. Methods: Samples were collected from totally, 10 animals (3 cattle, 3 goats and 4 sheep) with multiple papillomatosis lesions. Results: Clinically, exophytic multiple, cauliflower-like growths (warts) of varying sizes (0.5-11 cm) were found in different areas of the animal’s bodies. Histopathological features were various degrees of koilocytosis, ortho and parakeratotic, hyperkeratosis, hypergranulosis in granular layer and acanthosis. Immunohistochemical (IHC) investigations revealed some nuclei in the granular and basal layers of the epidermis with intense positivity for papillomavirus antigen. All tumor samples were positive for p53 expression that appeared as a strong cytoplasmic and perinuclear staining mainly in the basal and parabasal layers. Conclusion: this study described the papillomavirus lesions in bovine, ovine and caprine, that located in different anatomical areas with minor variations in histopathological features. The tumor samples showed positive results for PV antigen and P53 expression that considered as the useful markers in the diagnosis of cutaneous papilloma.