Retinoblastoma Binding Protein Research Articles

Rbbp6-Mediated Bmal1 Ubiquitination Inhibits YAP1 Signaling Pathway to Promote Ferroptosis in Diabetes-Induced Testicular Damage.

Diabetes-induced testicular damage (DITD) is a common complication of diabetes. We investigated underlying mechanism of retinoblastoma-binding protein 6 (Rbbp6)-mediated brain and muscle ARNT-like 1 (Bmal1) ubiquitination in modulating ferroptosis in DITD. Spermatogenic cell apoptosis and viability were measured by flow cytometry and cell counting kit 8 (CCK-8), respectively. The impact of Rbbp6 and Bmal1 on ferroptosis was assessed by determining expression of ferroptosis markers glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11), and levels of malondialdehyde (MDA), glutathione (GSH), iron, and lipid peroxidation. Co-immunoprecipitation was performed to determine the interaction between Rbbp6 and Bmal1, as well as the ubiquitination level of Bmal1. The expression levels of Rbbp6, Bmal1, Yes-associated protein 1 (YAP1), ferroptosis markers, and testicular steroidogenic enzymes were tested by Western blot. Bmal1 protein expression was significantly downregulated, while Rbbp6 was upregulated in DITD mouse model and high glucose (HG)-induced GC-1 spg cells. Overexpression of Bmal1 improved testicular injury in diabetic mice, reduced 4-hydroxynonenal (4-HNE), MDA, iron levels, and increased expression levels of GPX4, SLC7A11, GSH, as well as testicular steroidogenic enzymes. Rbbp6 decreased Bmal1 level through promoting its ubiquitination. Meanwhile, Rbbp6 knockdown inhibited the ferroptosis of HG-induced GC-1 spg cells, which were abolished by silencing Bmal1. In addition, knockdown of YAP1 or treatment with ferroptosis inducer erastin blocked the above effects caused by Bmal1 overexpression. Rbbp6-mediated Bmal1 ubiquitination suppressed YAP1 pathway, promoting ferroptosis in DITD. This study highlighted Rbbp6/Bmal1/YAP1 axis as a potential therapeutic target for mitigating DITD.

RBBP6-Mediated ERRα Degradation Contributes to Mitochondrial Injury in Renal Tubular Cells in Diabetic Kidney Disease.

Diabetic Kidney Disease (DKD), a major precursor to end-stage renal disease, involves mitochondrial dysfunction in proximal renal tubular cells (PTCs), contributing to its pathogenesis. Estrogen-related receptor α (ERRα) is essential for mitochondrial integrity in PTCs, yet its regulation in DKD is poorly understood. This study investigates ERRα expression and its regulatory mechanisms in DKD, assessing its therapeutic potential. Using genetic, biochemical, and cellular approaches, ERRα expression Was examined in human DKD specimens and DKD mouse models. We identified the E3 ubiquitin ligase retinoblastoma binding protein 6 (RBBP6) as a regulator of ERRα, promoting its degradation through K48-linked polyubiquitination at the K100 residue. This degradation pathway significantly contributed to mitochondrial injury in PTCs of DKD models. Notably, conditional ERRα overexpression or RBBP6 inhibition markedly reduced mitochondrial damage in diabetic mice, highlighting ERRα's protective role in maintaining mitochondrial integrity. The interaction between RBBP6 and ERRα opens new therapeutic avenues, suggesting that modulating RBBP6-ERRα interactions could be a strategy for preserving mitochondrial function and slowing DKD progression.

The role of lipoprotein‑associated phospholipase A2 in inflammatory response and macrophage infiltration in sepsis and the regulatory mechanisms.

Lipoproteinassociated phospholipase A2 (Lp-PLA2), encoded by the phospholipase A2 group VII (Pla2g7) gene, has been pertinent to inflammatory responses. This study investigates the correlation between Lp-PLA2 and inflammatory injury in septic mice and explores its regulatory mechanism. Lp-PLA2 was found to be upregulated in the serum of septic mice induced by cecal ligation and puncture and in the culture supernatant of RAW264.7 cells following lipopolysaccharide and adenosine triphosphate treatments. The contents of Lp-PLA2 were positively correlated with increased concentrations of proinflammatory cytokines in patients with sepsis. Both animal and cellular models showed increased concentrations of proinflammatory cytokines. Spi-1 proto-oncogene (Spi1), highly expressed in these models, was found to activate Pla2g7 transcription. Knockdown of Pla2g7 or Spi1 reduced the proinflammatory cytokine production, mitigated organ damage in mice, and suppressed macrophage migration in vitro. Retinoblastoma binding protein 6 (Rbbp6), poorly expressed in both models, was found to reduce Spi1 protein stability through ubiquitination modification. Rbbp6 overexpression similarly suppressed inflammatory activation of RAW264.7 cells, which was counteracted by Pla2g7 or Spi1 upregulation. In summary, this study demonstrates that the Pla2g7 loss and Spi1 upregulation participate in inflammatory responses in sepsis by elevating the Lp-PLA2 levels.

Probing the Effects of Retinoblastoma Binding Protein 6 (RBBP6) Knockdown on the Sensitivity of Cisplatin in Cervical Cancer Cells.

Cervical cancer is a major cause of death in women despite the advancement of current treatment modalities. The conventional therapeutic agent, cisplatin (CCDP), is the standard treatment for CC; however, resistance often develops due to the cancer's heterogeneity. Therefore, a detailed elucidation of the specific molecular mechanisms driving CC is crucial for the development of targeted therapeutic strategies. Retinoblastoma binding protein 6 (RBBP6) is a potential biomarker associated with cell proliferation and is upregulated in cervical cancer sites, exhibiting apoptosis and dysregulated p53 expression. Furthermore, RBBP6 has been demonstrated to sensitize cancer cells to radiation and certain chemotherapeutic agents by regulating the Bcl-2 gene, thus suggesting a crosstalk among RBBP6/p53/BCL-2 oncogenic signatures. The present study, therefore, investigated the relationship between cisplatin and RBBP6 expression in CC cells. Herein, we first explored bioinformatics simulations and identified that the RBBP6/p53/BCL-2 signaling pathway is overexpressed and correlated with CC. For further analysis, we explored the Genomics of Drug Sensitivity in Cancer (GDSC) and found that most of the CC cell lines are sensitive to CCDP. To validate these findings, RBBP6 was silenced in HeLa and Vero cells using RNAi technology, followed by measurement of wild-type p53 and Bcl-2 at the mRNA level using qPCR. Cells co-treated with cisplatin and siRBBP6 were subsequently analyzed for apoptosis induction and real-time growth monitoring using flow cytometry and the xCELLigence system, respectively. Cancer cells in the co-treatment group showed a reduction in apoptosis compared to the cisplatin-treated group. Moreover, the real-time growth monitoring revealed a reduced growth rate in RBBP6 knockdown cells treated with cisplatin. Although wild-type p53 remained unchanged in the co-treatment group of cancer cells, Bcl-2 was completely repressed, suggesting that RBBP6 is necessary for sensitizing cervical cancer cells to cisplatin treatment by downregulating Bcl-2. The Vero cell population, which served as a non-cancerous control cell line in this study, remained viable following treatment with both siRBBP6 and cisplatin. Findings from this study suggest that RBBP6 expression promotes cisplatin sensitivity in HeLa cells through Bcl-2 downregulation. Knockdown of RBBP6 limits apoptosis induction and delays cell growth inhibition in response to cisplatin. The knowledge obtained here has the potential to help improve cisplatin efficacy through personalized administration based on the expression profile of RBBP6 among individual patients.

TSPAN8+ myofibroblastic cancer-associated fibroblasts promote chemoresistance in patients with breast cancer.

Cancer-associated fibroblasts (CAFs) are abundant stromal cells in the tumor microenvironment that promote cancer progression and relapse. However, the heterogeneity and regulatory roles of CAFs underlying chemoresistance remain largely unclear. Here, we performed a single-cell analysis using high-dimensional flow cytometry analysis and identified a distinct senescence-like tetraspanin-8 (TSPAN8)+ myofibroblastic CAF (myCAF) subset, which is correlated with therapeutic resistance and poor survival in multiple cohorts of patients with breast cancer (BC). TSPAN8+ myCAFs potentiate the stemness of the surrounding BC cells through secretion of senescence-associated secretory phenotype (SASP)-related factors IL-6 and IL-8 to counteract chemotherapy. NAD-dependent protein deacetylase sirtuin 6 (SIRT6) reduction was responsible for the senescence-like phenotype and tumor-promoting role of TSPAN8+ myCAFs. Mechanistically, TSPAN8 promoted the phosphorylation of ubiquitin E3 ligase retinoblastoma binding protein 6 (RBBP6) at Ser772 by recruiting MAPK11, thereby inducing SIRT6 protein destruction. In turn, SIRT6 down-regulation up-regulated GLS1 and PYCR1, which caused TSPAN8+ myCAFs to secrete aspartate and proline, and therefore proved a nutritional niche to support BC outgrowth. By demonstrating that TSPAN8+SIRT6low myCAFs were tightly associated with unfavorable disease outcomes, we proposed that the combined regimen of anti-TSPAN8 antibody and SIRT6 activator MDL-800 is a promising approach to overcome chemoresistance. These findings highlight that senescence contributes to CAF heterogeneity and chemoresistance and suggest that targeting TSPAN8+ myCAFs is a promising approach to circumvent chemoresistance.

Abstract 1440: Retinoblastoma binding protein 9 regulates intestinal inflammation and inflammation-associated carcinogenesis

Abstract The intestinal epithelium constitutes the mucosal barrier, and its dysfunction promotes microbial invasion from the gut lumen, which further induces the intestinal inflammation. Chronic intestinal inflammation, as observed in poorly controlled inflammatory bowel diseases (IBD), leads to the development of inflammation-associated cancer. Thus, a deeper understanding of the regulatory system that controls intestinal homeostasis is essential. Retinoblastoma-binding protein 9 (RBBP9) is expressed in a range of human cancer cells. RBBP9 was initially reported to have the ability to regulate the cell cycle pathway. More recent studies have identified RBBP9 as a serine hydrolase (SH) that promotes cancer progression by subverting the anti-proliferative function of TGFβ in a pancreatic cancer model. However, little is known about the roles RBBP9 plays in the development of intestinal cancer. In this study, we investigated the role of RBBP9 in inflammation and inflammation-associated cancer in the intestine. Analyses of surgically resected human samples and publicly available datasets demonstrated that RBBP9 expression was reduced in patient samples of ulcerative colitis (UC) and colitis-associated cancer (CAC). Genetically knockout mice for Rbbp9 (Rbbp9−/−) were susceptible to experimental colitis induced by dextran sulfate sodium (DSS). Furthermore, Rbbp9−/− mice exhibited enhanced azoxymethane/DSS-induced tumorigenesis, a model for human CAC. Mechanistically, RNA sequencing of these tumors showed increased enrichment of inflammation-associated signatures, including interferon signaling pathways. Ex vivo organoid experiments revealed the suppressive function of RBBP9 in interferon signaling-mediated apoptosis of the intestinal epithelium. Simultaneous inhibition of RBBP9 and STAT1, a downstream transcription factor of interferon signaling, reverted the apoptotic phenotype of Rbbp9−/− organoids. Collectively, our data provide insight into the understanding of the crucial role of RBBP9 in protecting against intestinal inflammation and inflammation-related tumorigenesis via regulating the interferon signaling-mediated apoptotic pathway. Citation Format: Yuki Nakanishi, Kensuke Hamada, Mayuki Omatsu, Kosuke Iwane, Hiroki Kitamoto, Maokoto Okabe, Yu Muta, Shuji Yamamoto, Akihisa Fukuda, Hiroaki Kasashima, Hiroshi Seno. Retinoblastoma binding protein 9 regulates intestinal inflammation and inflammation-associated carcinogenesis [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 1440.

Development of Complementary Photo-arginine/lysine to Promote Discovery of Arg/Lys hPTMs Interactomes.

Arginine and lysine, frequently appearing as a pair on histones, have been proven to carry diverse modifications and execute various epigenetic regulatory functions. However, the most context-specific and transient effectors of these marks, while significant, have evaded study as detection methods have thus far not reached a standard to capture these ephemeral events. Herein, a pair of complementary photo-arginine/δ-photo-lysine (R-dz/K-dz) probes is developed and involve these into histone peptide, nucleosome, and chromatin substrates to capture and explore the interactomes of Arg and Lys hPTMs. By means of these developed tools, this study identifies that H3R2me2a can recruit MutS protein homolog 6 (MSH6), otherwise repelDouble PHD fingers 2 (DPF2), Retinoblastoma binding protein 4/7 (RBBP4/7). And it is disclosed that H3R2me2a inhibits the chromatin remodeling activity of the cBAF complex by blocking the interaction between DPF2 (one component of cBAF) and the nucleosome. In addition, the novel pairs of H4K5 PTMs and respective readers are highlighted, namely H4K5me-Lethal(3)malignant brain tumor-like protein 2 (L3MBTL2), H4K5me2-L3MBTL2, and H4K5acK8ac-YEATS domain-containing protein 4 (YEATS4). These powerful tools pave the way for future investigation of related epigenetic mechanisms including but not limited to hPTMs.

Overlapping characteristics of weak interactions of two transcriptional regulators with WDR5

The WD40 repeat protein 5 (WDR5) is a nuclear hub that critically influences gene expression by interacting with transcriptional regulators. Utilizing the WDR5 binding motif (WBM) site, WDR5 interacts with the myelocytomatosis (MYC), an oncoprotein transcription factor, and the retinoblastoma-binding protein 5 (RbBP5), a scaffolding element of an epigenetic complex. Given the clinical significance of these protein-protein interactions (PPIs), there is a pressing necessity for a quantitative assessment of these processes. Here, we use biolayer interferometry (BLI) to examine interactions of WDR5 with consensus peptide ligands of MYC and RbBP5. We found that both interactions exhibit relatively weak affinities arising from a fast dissociation process. Remarkably, live-cell imaging identified distinctive WDR5 localizations in the absence and presence of full-length binding partners. Although WDR5 tends to accumulate within nucleoli, WBM-mediated interactions with MYC and RbBP5 require their localization outside nucleoli. We utilize fluorescence resonance energy transfer (FRET) microscopy to confirm these weak interactions through a low FRET efficiency of the MYC-WDR5 and RbBP5-WDR5 complexes in living cells. In addition, we evaluate the impact of peptide and small-molecule inhibitors on these interactions. These outcomes form a fundamental basis for further developments to clarify the multitasking role of the WBM binding site of WDR5.

Ropivacaine inhibits the malignant behavior of lung cancer cells by regulating retinoblastoma-binding protein 4.

Ropivacaine is a local anesthetic commonly used in regional nerve blocks to manage perioperative pain during lung cancer surgery. Recently, the antitumor potential of ropivacaine has received considerable attention. Our previous study showed that ropivacaine treatment inhibits the malignant behavior of lung cancer cells in vitro. However, the potential targets of ropivacaine in lung cancer cells have not yet been fully identified. This study aimed to explore the antitumor effects and mechanisms of action of ropivacaine in lung cancer. Lung cancer A549 cells were treated with or without 1 mM ropivacaine for 48 h. Quantitative proteomics was performed to identify the differentially expressed proteins (DEPs) triggered by ropivacaine treatment. STRING and Cytoscape were used to construct protein-protein interaction (PPI) networks and analyze the most significant hub genes. Overexpression plasmids and small interfering RNA were used to modulate the expression of key DEPs in A549 and H1299 cells. MTS, transwell assays, and flow cytometry were performed to determine whether the key DEPs were closely related to the anticancer effect of ropivacaine on the malignant behavior of A549 and H1299 cells. Quantitative proteomic analysis identified 327 DEPs (185 upregulated and 142 downregulated proteins) following ropivacaine treatment. Retinoblastoma-binding protein 4 (RBBP4) was one of the downregulated DEPs and was selected as the hub protein. TCGA database showed that RBBP4 was significantly upregulated in lung cancer and was associated with poor patient prognosis. Inhibition of RBBP4 by siRNA resulted in a significant decrease in the proliferation and invasive capacity of lung cancer cells and the induction of cell cycle arrest. Additionally, the results indicated RBBP4 knockdown enhanced antitumor effect of ropivacaine on A549 and H1299 cells. Conversely, the overexpression of RBBP4 using plasmids reversed the inhibitory effects of ropivacaine. Our data suggest that ropivacaine suppresses lung cancer cell malignancy by downregulating RBBP4 protein expression, which may help clarify the mechanisms underlying the antitumor effects of ropivacaine.

Unveiling the molecular structure and role of RBBP4/7: implications for epigenetic regulation and cancer research.

Retinoblastoma-binding protein (RBBP) family is a class of proteins that can interact with tumor suppressor retinoblastoma protein (pRb). RBBP4 and RBBP7 are the only pair of homologous proteins in this family, serving as scaffold proteins whose main function is to offer a platform to indirectly connect two proteins. This characteristic allows them to extensively participate in the binding of various proteins and epigenetic complexes, indirectly influencing the function of effector proteins. As a result, they are often highlighted in organism activities involving active epigenetic modifications, such as embryonic development and cancer activation. In this review, we summarize the structural characteristics of RBBP4/7, the complexes they are involved in, their roles in embryonic development and cancer, as well as potential future research directions, which we hope to inspire the field of epigenetic research in the future.

X-linked RBBP7 mutation causes maturation arrest and testicular tumors.

Maturation arrest (MA) is a subtype of non-obstructive azoospermia, and male infertility is a known risk factor for testicular tumors. However, the genetic basis for many affected individuals remains unknown. Here, we identified a deleterious hemizygous variant of X-linked retinoblastoma-binding protein 7 (RBBP7) as a potential key cause of MA, which was also found to be associated with the development of Leydig cell tumors. This mutation resulted in premature protein translation termination, affecting the sixth WD40 domain of the RBBP7 and the interaction of the mutated RBBP7 with histone H4. Decreased BRCA1 and increased γH2AX were observed in the proband. In mouse spermatogonial and pachytene spermatocyte-derived cells, deprivation of rbbp7 led to cell cycle arrest and apoptosis. In Drosophila, knockdown of RBBP7/Caf1-55 in germ cells resulted in complete absence of germ cells and reduced testis size, whereas knockdown of RBBP7/Caf1-55 in cyst cells resulted in hyperproliferative testicular cells. Interestingly, male infertility caused by Caf1-55 deficiency was rescued by ectopic expression of wild-type human RBBP7 but not mutant variants, suggesting the importance of RBBP7 in spermatogenesis. Our study provides insights into the mechanisms underlying the co-occurrence of MA and testicular tumors and may pave the way for innovative genetic diagnostics of these 2 diseases.

Expression and clinical significance of RBBP4 gene in lower-grade glioma: An integrative analysis

This study investigated the expression pattern of retinoblastoma binding protein 4 (RBBP4) gene in glioma and explored its associations with clinicopathologic characteristics and prognosis of patients. Data retrieved from the GEPIA, CGGA, HPA and TIMER databases were processed to analyze RBBP4 expression in glioma and investigate its relationship with clinicopathologic characteristics, tumor immune infiltration and prognosis in glioma patients. Immunohistochemistry was applied to determine the expression of RBBP4 protein in glioma tissue. Additionally, the Coexpedia database was visited to identify co-expressed genes for RBBP4 gene, while the Cytoscape software was run to visualize the enriched GO entries and KEGG pathways of these co-expressed genes. The expression levels of RBBP4 in lower-grade glioma (LGG) and glioblastoma (GBM) tissues were markedly elevated when compared to normal tissues (both p < 0.05). The up-regulation of RBBP4 expression was associated with an increase in WHO grade (II-IV), wild-type IDH, and 1p/19q non-codeletion (all p < 0.05). Multi-variate Cox regression analysis showed that both increased abundance of infiltrating macrophages and up-regulated RBBP4 expression independently predicted poor survival outcomes in LGG patients (both p < 0.05). Furthermore, RBBP4 expression exhibited significant positive correlations with the abundance of infiltrating B cell, CD8+ T cell, CD4+ T cell, macrophage, neutrophil, and dendritic cell in LGG (all p < 0.05). Functional enrichment analyses indicated that the co-expressed genes associated with RBBP4 were highly involved in pathways such as the p53 signaling pathway, cell cycle, DNA replication, glutathione metabolism, as well as biological processes including cell cycle process, DNA replication, and DNA repair. High levels of RBBP4 are predictive for the poor survival outcome of LGG patients. RBBP4 gene, therefore, is expected to be a potential biomarker for prognosis of LGG and a target for immunotherapy.

TIPS-07 LONGITUDINAL CSF PROTEOMICS FOR MONITORING AND PHARMACODYNAMIC BIOMARKER DISCOVERY

Abstract Rapid feedback is needed to understand the individualized biological impacts of novel glioma therapies. We are performing glioma biomarker discovery by serial cerebrospinal fluid (CSF) sampling from Ommaya reservoirs or lumbar punctures to determine how the CSF proteome can reveal early longitudinal intelligence regarding glioma status, biology, and therapeutic response. We aimed to leverage access to each patient’s glioma via CSF to identify candidate biomarkers of tumor burden, biology, and therapeutic challenge. Global proteomic CSF analysis was performed via Somalogic– an aptamer-based technology for highly sensitive and specific analysis of over 7,000 proteins. Discovery analysis comprised of the top 500 ranked proteins in the CSF from seven patients with high-grade gliomas versus non-glioma (normal pressure hydrocephalus) controls. The top HGG proteins were then filtered to include only proteins that met two additional criteria of 1) decrease with resection, and 2) increase with recurrence in individual paired patient samples. Additionally, in individual patients for whom serial CSF samples were available, protein fold change lists were created and ranked to determine the most or least differentially abundant proteins between various time points. Proteomic enrichment analysis revealed a conserved HGG CSF proteomic signature defined by 79 proteins, including ones known to be over-expressed in solid tumor malignancies, such as retinoblastoma binding protein 4, heat shock protein 90, and sorcin. The HGG proteomic signature was consistently enriched in an independent validation cohort consisting of 13 gliomas diverse in primary versus recurrent status, subtype, and grade, when compared to independent control CSF samples. Encouragingly, proteins in the HGG signature decreased in two patients following decreased tumor burden from surgical resection. Our preliminary data demonstrate the ability to gain detailed, individualized insights regarding glioma biology, tumor burden, and evolution through global CSF proteomics acquired from longitudinal neurosurgical access to each patient’s unique glioma.

Abstract 4925: Cannabidiol and Cannabis Sativa as a potential treatment in vitro prostate cancer cells silenced with RBBp6 and PC3 xenograft

Abstract Background: Prostate cancer is the second most frequently occurring carcinoma in males worldwide and one of the leading causes of death in men around the world. Recent studies estimate that over 1.4 million males are diagnosed with prostate cancer on an annual basis, with approximately 375 000 succumbing to the disease annually. With current treatments continuing to show severe side effects, there is a need for new treatments. In this study we looked at the effect of cannabis sativa extract, cannabidiol and cisplatin on prostate cancer cells, PC3. Methods: In addressing the above questions, we employed the MTT assay to measure the antiproliferative effect on PC3 cells following treatment with varying concentrations of Cannabis sativa extract, cisplatin and cannabidiol. xCELLigence was also used to confirm the IC50 activity in which cells were grown in a 16 well plate coated with gold and monitor cell attachment. Caspase 3/7 activity was also measured using 96 well-plate following treatment. Western-blot and qRT-PCR was also used to measure the gene expression of tumor suppressor genes, p53, Bax and Bcl2. Animal studies were employed to measure the growth of PC3-mouse derived cancer to evaluate the effect of compounds in vivo. Results: From the treatment with varying concentrations of Cannabis sativa extract, cannabidiol and cisplatin, we have observed that the three compounds induced antiproliferation of PC3 cancer cell lines through the activation of caspase 3/7 activity. We also observed induction of apoptosis in these cells following silencing of retinoblastoma binding protein 6 (RBBP6), with upregulation of p53 and bax mRNA expression, and a reduction in Bcl2 gene expression. The growth of tumors in the mouse models were reduced following treatment with cisplatin and cannabidiol. Conclusion: We demonstrated that cannabidiol is a viable therapy to treat prostate cancer cells, in combination with silencing of RBBP6. This suggests that cannabidiol rather Cannabis sativa extract may play an important role in reducing cancer progression. Citation Format: Lesetja Raymond Motadi, Zodwa E. Jantjies. Cannabidiol and Cannabis Sativa as a potential treatment in vitro prostate cancer cells silenced with RBBp6 and PC3 xenograft. [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 4925.

CircAGO2 promotes colorectal cancer progression by inhibiting heat shock protein family B (small) member 8 via miR-1-3p/retinoblastoma binding protein 4 axis.

This paper was to uncover the mechanism of circular RNA Argonaute 2 (circAGO2) in colorectal cancer (CRC) progression. The expression of circAGO2 was detected in CRC cells and tissues, and the relationship between clinicopathological features of CRC and circAGO2 level was evaluated. The growth and invasion of CRC cells and subcutaneous xenograft of nude mice were measured to evaluate the effect of circAGO2 on CRC development. Bioinformatics databases were applied to analyze levels of retinoblastoma binding protein 4 (RBBP4) and heat shock protein family B 8 (HSPB8) in cancer tissues. The relevance of circAGO2 and RBBP4 expression and the relationship between RBBP4 and HSPB8 during histone acetylation were assessed. The targeting relationship between miR-1-3p and circAGO2 or RBBP4 was predicted and confirmed. The effects of miR-1-3p and RBBP4 on biological functions of CRC cells were also verified. CircAGO2 was upregulated in CRC. CircAGO2 promoted the growth and invasion of CRC cells. CircAGO2 competitively bound to miR-1-3p and regulated RBBP4 expression, thus inhibiting HSPB8 transcription by promoting histone deacetylation. Silencing circAGO2 enhanced miR-1-3p expression and reduced RBBP4 expression, while suppression of miR-1-3p downgraded levels of miR-1-3p, up-regulated RBBP4, and facilitated cell proliferation and invasion in the presence of silencing circAGO2. RBBP4 silencing decreased RBBP4 expression and reduced proliferation and invasion of cells where circAGO2 and miR-1-3p were silenced. CircAGO2 overexpression decoyed miR-1-3p to increase RBBP4 expression, which inhibited HSPB8 transcription via histone deacetylation in HSPB8 promoter region, promoting proliferation and invasion of CRC cells.

Cannabidiol and Cannabis Sativa as a potential treatment in vitro prostate cancer cells silenced with RBBp6 and PC3 xenograft

BackgroundProstate cancer is the second most frequently occurring carcinoma in males worldwide and one of the leading causes of death in men around the world. Recent studies estimate that over 1.4 million males are diagnosed with prostate cancer on an annual basis, with approximately 375,000 succumbing to the disease annually. With current treatments continuing to show severe side effects, there is a need for new treatments. In this study we looked at the effect of cannabis sativa extract, cannabidiol and cisplatin on prostate cancer cells, PC3.MethodsIn addressing the above questions, we employed the MTT assay to measure the antiproliferative effect on PC3 cells following treatment with varying concentrations of Cannabis sativa extract, cisplatin and cannabidiol. xCELLigence was also used to confirm the IC50 activity in which cells were grown in a 16 well plate coated with gold and monitor cell attachment. Caspase 3/7 activity was also measured using 96 well-plate following treatment. Western-blot and qRT-PCR was also used to measure the gene expression of tumour suppressor genes, p53, Bax and Bcl2. Animal studies were employed to measure the growth of PC3-mouse derived cancer to evaluate the effect of compounds in vivo.ResultsFrom the treatment with varying concentrations of Cannabis sativa extract, cannabidiol and cisplatin, we have observed that the three compounds induced antiproliferation of PC3 cancer cell lines through the activation of caspase 3/7 activity. We also observed induction of apoptosis in these cells following silencing of retinoblastoma binding protein 6 (RBBP6), with upregulation of p53 and bax mRNA expression, and a reduction in Bcl2 gene expression. The growth of tumours in the mouse models were reduced following treatment with cisplatin and cannabidiol.ConclusionWe demonstrated that cannabidiol is a viable therapy to treat prostate cancer cells, in combination with silencing of RBBP6. This suggests that cannabidiol rather Cannabis sativa extract may play an important role in reducing cancer progression.

Retinoblastoma-Binding Protein 5 Regulates H3K4 Methylation Modification to Inhibit the Proliferation of Melanoma Cells by Inactivating the Wnt/β-Catenin and Epithelial-Mesenchymal Transition Pathways.

Histone 3 lysine 4 methylation (H3K4me), especially histone 3 lysine 4 trimethylation (H3K4me3), is one of the most extensively studied patterns of histone modification and plays crucial roles in many biological processes. However, as a part of H3K4 methyltransferase that participates in H3K4 methylation and transcriptional regulation, retinoblastoma-binding protein 5 (RBBP5) has not been well studied in melanoma. The present study sought to explore RBBP5-mediated H3K4 histone modification and the potential mechanisms in melanoma. RBBP5 expression in melanoma and nevi specimens was detected by immunohistochemistry. Western blotting was performed for three pairs of melanoma cancer tissues and nevi tissues. In vitro and in vivo assays were used to investigate the function of RBBP5. The molecular mechanism was determined using RT-qPCR, western blotting, ChIP assays, and Co-IP assays. Our study showed that RBBP5 was significantly downregulated in melanoma tissue and cells compared with nevi tissues and normal epithelia cells (P < 0.05). Reducing RBBP5 in human melanoma cells leads to H3K4me3 downregulation and promotes cell proliferation, migration, and invasion. On the one hand, we verified that WSB2 was an upstream gene of RBBP5-mediated H3K4 modification, which could directly bind to RBBP5 and negatively regulate its expression. On the other hand, we also confirmed that p16 (a cancer suppressor gene) was a downstream target of H3K4me3, the promoter of which can directly bind to H3K4me3. Mechanistically, our data revealed that RBBP5 inactivated the Wnt/β-catenin and epithelial-mesenchymal transition (EMT) pathways (P < 0.05), leading to melanoma suppression. Histone methylation is rising as an important factor affecting tumorigenicity and tumor progression. Our findings verified the significance of RBBP5-mediated H3K4 modification in melanoma and the potential regulatory mechanisms of melanoma proliferation and growth, suggesting that RBBP5 is a potential therapeutic target for the treatment of melanoma.

Expression, purification, and characterisation of the p53 binding domain of Retinoblastoma binding protein 6 (RBBP6).

RBBP6 is a 250 kDa eukaryotic protein known to be a negative regulator of p53 and essential for embryonic development. Furthermore, RBBP6 is a critical element in carcinogenesis and has been identified as a potential biomarker for certain cancers. RBBP6's ability to interact with p53 and cause its degradation makes it a potential drug target in cancer therapy. Therefore, a better understating of the p53 binding domain of RBBP6 is needed. This study presents a three-part purification protocol for the polyhistidine-tagged p53 binding domain of RBBP6, expressed in Escherichia coli bacterial cells. The purified recombinant domain was shown to have structure and is functional as it could bind endogenous p53. We characterized it using clear native PAGE and far-UV CD and found that it exists in a single form, most likely monomer. We predict that its secondary structure is predominantly random coil with 19% alpha-helices, 9% beta-strand and 14% turns. When we exposed the recombinant domain to increasing temperature or known denaturants, our investigation suggested that the domain undergoes relatively small structural changes, especially with increased temperature. Moreover, we notice a high percentage recovery after returning the domain close to starting conditions. The outcome of this study is a pure, stable, and functional recombinant RBBP6-p53BD that is primarily intrinsically disordered.

KTN1-AS1, a SOX2-mediated lncRNA, activates epithelial–mesenchymal transition process in esophageal squamous cell carcinoma

Kinectin 1 antisense RNA 1 (KTN1-AS1), a long non-coding RNA (lncRNA), has been proved to have tumor-promoting properties and its expression is enhanced in several human tumors. However, the role of KTN1-AS1 in the pathogenesis of esophageal squamous cell carcinoma (ESCC) remains unknown. This study aimed to investigate the expression status, functional roles, and molecular mechanisms of KTN1-AS1 in the development of ESCC. Considerable upregulation of KTN1-AS1 was confirmed in esophageal cancer cells and ESCC tissues and its expression was associated with TNM stage, pathological differentiation, and lymph node metastasis. SOX2 directly activated transcription of KTN1-AS1, and overexpression of KTN1-AS1 facilitated ESCC cells proliferation and invasion in vitro and in vivo. Furthermore, KTN1-AS1 could bind to retinoblastoma binding protein 4 (RBBP4) in the nucleus and enhanced its binding with histone deacetylase 1 (HDAC1), thereby activating the epithelial–mesenchymal transition (EMT) process through downregulating E-cadherin expression at the epigenetic level. In conclusion, KTN1-AS1, induced by SOX2, acts as a tumor-promoting gene and may serve as a potential therapeutic and prognostic biomarker for ESCC.

Glioma Espionage From Longitudinal CSF Proteomics

Abstract AIMS Rapid, detailed feedback is needed to understand the individualized biological impacts of novel glioma therapies. We are performing glioma biomarker discovery by serial cerebrospinal fluid (CSF) sampling from Ommaya reservoirs to determine how the CSF proteome can reveal early longitudinal intelligence regarding glioma status, biology, and therapeutic response. METHOD Global proteomic analysis of CSF was performed on the Somalogic platform – an aptamer-based technology for highly sensitive and specific analysis of over 7,000 proteins. Discovery analysis comprised of the top-500 ranked proteins in CSF from seven patients with high-grade gliomas (HGG) versus non-glioma controls. The top-500 HGG proteins were then preliminarily filtered to include only proteins that met two additional criteria of decrease with resection and increase with recurrence in individual paired patient samples. RESULTS Proteomic enrichment analysis revealed a conserved HGG CSF proteomic signature defined by 79 proteins, including ones known to be over-expressed in solid tumor malignancies, such as retinoblastoma binding protein 4, heat shock protein 90, and sorcin. The HGG proteomic signature was consistently enriched in an independent validation cohort consisting of 13 gliomas diverse in primary versus recurrent status, subtype, and grade, when compared to control CSF samples. Encouragingly, proteins in the HGG signature decreased in the two patients for whom CSF was collected prior to and after resection (both at POD16 and POD18) with decreased tumor burden. CONCLUSION Our data demonstrate the ability to gain detailed, individualized insights regarding glioma biology, tumor burden, and evolution through global CSF proteomics acquired from longitudinal access to gliomas via Ommaya reservoirs.