Ring Finger Protein 43 Research Articles

Abstract B090: Evaluating the molecular determinants of PDAC racial health disparities

Abstract Pancreatic cancer is the 3rd leading cause of cancer-related deaths, with a 5-year survival rate of just 13%. Pancreatic ductal adenocarcinoma (PDAC), accounting for over 90% of pancreatic malignancies, has the highest incidence rate, mortality rate, and shortest survival times in non- Hispanic Black (defined here as African and/or African American ancestry) patients compared to other races. To identify molecular features that may contribute to racial health disparities in PDAC, we used quantitative mass spectrometry to determine the proteomic signatures unique to tumor racial origin in a cohort of 30 Caucasian and 12 African American patients. This analysis of the bulk tumor proteome revealed 183 proteins were differentially expressed between these groups. The protein with the highest expression in tumors from Black patients relative to Whites was Ring Finger Protein 2 (RNF2) that acts as an epigenetic transcriptional repressor in developmental processes pertaining to cellular differentiation. To determine the molecular pathways in PDAC regulated by RNF2, chromatin immunoprecipitation and sequencing (ChIP-seq) was performed on MIA-PaCa2 and a primary PDAC cell line derived from a Black patient to identify binding sites for RNF2 in PDAC cells. From the 1,142 replicated peaks identified, RNF2 peaks were localized over the transcription start site of 644 unique transcripts, including GATA6, which is an important determinant in PDAC subtype delineation with higher GATA6 expression associated with the less aggressive and more chemosensitive Classical subtype. Modulating RNF2 levels/activity by exogenous expression, short hairpin RNA (shRNA) knockdown, and small molecule inhibition confirms that GATA6 is a target of RNF2 repression in PDAC. Multi-omic (transcriptomic, proteomic, and phosphoproteomic) characterization was performed on MIA-PaCa2 cells with CRISPR-Cas9 knockout (KO) of RNF2. Differential gene expression (DE) analysis revealed changes in platinum drug resistance, EMT, and innate immune response, which are characteristic of the aggressive Inflammatory PDAC subtype we previously reported using proteomics. Total and phospho-proteomics corroborated these results identifying changes in DNA repair, inflammation response, TNFα mediated signaling, and apoptotic signaling. These results reflect the enrichment of Inflammatory subtype markers in tumors originating in Black patients. In agreement with these results, RNF2 KO shows a significant reduction in migration, as well as an increase in sensitivity to oxaliplatin. Our work identifies differences in the underlying proteome of PDAC tumors associated with tumor racial origin and describes how elevated expression of RNF2 in PDACs from Black patients contribute to inflammation and subtype delineation. This study delineates the associations between tumor racial origin, subtype specification, and response to therapy that could mitigate PDAC health disparities and be broadly applied across all PDAC patients. Citation Format: Anthony E Johansen-Sallee, Alexa M Barber, Henry C.-H. Law, Vignesh Vudatha, Jose Trevino, Nicholas T Woods. Evaluating the molecular determinants of PDAC racial health disparities [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B090.

E3 ligase RNF2 inhibits porcine circovirus type 3 replication by targeting its capsid protein for ubiquitination-dependent degradation.

Porcine circovirus type 3 (PCV3) is closely associated with various diseases, such as the porcine dermatitis, nephropathy syndrome, and multisystemic clinicopathological diseases. PCV3-associated diseases are increasingly recognized as severe diseases in the global swine industry. Ring finger protein 2 (RNF2), an E3 ubiquitin ligase exclusively located in the nucleus, contributes to various biological processes. This ligase interacts with the PCV3 Cap. However, its role in PCV3 replication remains unclear. This study confirmed that the nuclear localization signal domain of the Cap and the RNF2 N-terminal RING domain facilitate the interaction between the Cap and RNF2. Furthermore, RNF2 promoted the binding of K48-linked polyubiquitination chains to lysine at positions 139 and 140 (K139 and K140) of the PCV3 Cap, thereby degrading the Cap. RNF2 knockdown and overexpression increased or decreased PCV3 replication, respectively. Moreover, the RING domain-deleted RNF2 mutant eliminated the RNF2-induced degradation of the PCV3 Cap and RNF2-mediated inhibition of viral replication. This indicates that both processes were associated with its E3 ligase activity. Our findings demonstrate that RNF2 can interact with and degrade the PCV3 Cap via its N-terminal RING domain in a ubiquitination-dependent manner, thereby inhibiting PCV3 replication.IMPORTANCEPorcine circovirus type 3 is a recently described pathogen that is prevalent worldwide, causing substantial economic losses to the swine industry. However, the mechanisms through which host proteins regulate its replication remain unclear. Here, we demonstrate that ring finger protein 2 inhibits porcine circovirus type 3 replication by interacting with and degrading the Cap of this pathogen in a ubiquitination-dependent manner, requiring its N-terminal RING domain. Ring finger protein 2-mediated degradation of the Cap relies on its E3 ligase activity and the simultaneous existence of K139 and K140 within the Cap. These findings reveal the mechanism by which this protein interacts with and degrades the Cap to inhibit porcine circovirus type 3 replication. This consequently provides novel insights into porcine circovirus type 3 pathogenesis and facilitates the development of preventative measures against this pathogen.

SMARCA2 and SMARCA4 Participate in DNA Damage Repair.

The switching/sucrose non-fermentable (SWI/SNF) Related, Matrix Associated, Actin Dependent Regulator Of Chromatin, Subfamily A (SMARCA) member 2 and member 4 (SMARCA2/4) are paralogs and act as the key enzymatic subunits in the SWI/SNF complex for chromatin remodeling. However, the role of SMARCA2/4 in DNA damage response remains unclear. Laser microirradiation assays were performed to examine the key domains of SMARCA2/4 for the relocation of the SWI/SNF complex to DNA lesions. To examine the key factors that mediate the recruitment of SMARCA2/4, the relocation of SMARCA2/4 to DNA lesions was examined in HeLa cells treated with inhibitors of Ataxia-telangiectasia-mutated (ATM), Ataxia telangiectasia and Rad3-related protein (ATR), CREB-binding protein (CBP) and its homologue p300 (p300/CBP), or Poly (ADP-ribose) polymerase (PARP) 1/2 as well as in H2AX-deficient HeLa cells. Moreover, by concomitantly suppressing SMARCA2/4 with the small molecule inhibitor FHD286 or Compound 14, the function of SMARCA2/4 in Radiation sensitive 51 (RAD51) foci formation and homologous recombination repair was examined. Finally, using a colony formation assay, the synergistic effect of PARP inhibitors and SMARCA2/4 inhibitors on the suppression of tumor cell growth was examined. We show that SMARCA2/4 relocate to DNA lesions in response to DNA damage, which requires their ATPase activities. Moreover, these ATPase activities are also required for the relocation of other subunits in the SWI/SNF complex to DNA lesions. Interestingly, the relocation of SMARCA2/4 is independent of γH2AX, ATM, ATR, p300/CBP, or PARP1/2, indicating that it may directly recognize DNA lesions as a DNA damage sensor. Lacking SMARCA2/4 prolongs the retention of γH2AX, Ring Finger Protein 8 (RNF8) and Breast cancer susceptibility gene 1 (BRCA1) at DNA lesions and impairs RAD51-dependent homologous recombination repair. Furthermore, the treatment of an SMARCA2/4 inhibitor sensitizes tumor cells to PARP inhibitor treatment. This study reveals SMARCA2/4 as a DNA damage repair factor for double-strand break repair.

M6A methylation of RNF43 inhibits the progression of endometriosis through regulating oxidative phosphorylation via NDUFS1.

Oxidative phosphorylation is becoming increasingly important in the induction and development of endometriosis. Recently, it has been reported that ring finger protein 43 (RNF43) is involved in the process of oxidative phosphorylation, but the mechanism remains unclear. Our investigation is to delve into the roles of RNF43 in endometriosis and elucidate the related mechanisms. We found RNF43 was downregulated in ectopic endometrial tissue and primary ectopic endometrial stromal cells (ECESCs). Knockdown of RNF43 enhanced cell viability and migration by activating oxidative phosphorylation in eutopic endometrial stromal cells (EUESCs), while overexpression of RNF43 led to the opposite results. Moreover, RNF43 reinforced the ubiquitination and degradation of NADH dehydrogenase Fe-S protein 1 (NDUFS1) by interacting with it. Likewise to RNF43 overexpression, NDUFS1 silencing inhibited cell viability, migration, and oxidative phosphorylation in ECESCs. NDUFS1 was a downstream target of RNF43, mediating its biological role in endometriosis. Interestingly, the expression and stability of RNF43 mRNA were regulated by the Methyltransferase-like 3 (METTL3)/IGF2BP2 m6A modification axis. The results of rat experiments showed decreased RNF43 expression and increased NDUFS1 expression in endometriosis rats, which was enhanced by METTL3 inhibition. Those observations indicated that m6A methylation-mediated RNF43 negatively affects viability and migration of endometrial stromal cells through regulating oxidative phosphorylation via NDUFS1. The discovery of METTL3/RNF43/NDUFS1 axis suggested promising therapeutic targets for endometriosis.

Hyperoside Inhibits RNF8-mediated Nuclear Translocation of β-catenin to Repress PD-L1 Expression and Prostate Cancer.

Hyperoside is a flavonol glycoside isolated from Hypericum perforatum L. that has inhibitory effects on cancer cells; however, its effects on prostate cancer (PCa) remain unclear. Therefore, we studied the anti-PCa effects of hyperoside and its underlying mechanisms in vitro and in vivo. This study aimed to explore the mechanism of hyperoside in anti-PCa. 3-(4,5-Dimethyl-2-Thiazolyl)-2,5-Diphenyl Tetrazolium Bromide (MTT), transwell, and flow cytometry assays were used to detect PCa cell growth, invasion, and cell apoptosis. Immunoblot analysis, immunofluorescence, immunoprecipitation, and quantitative real-time PCR (qRT-PCR) were used to analyze the antitumor mechanism of hyperoside. Hyperoside inhibited PCa cell growth, invasion, and cell cycle and induced cell apoptosis. Furthermore, RING finger protein 8 (RNF8), an E3 ligase that assembles K63 polyubiquitination chains, was predicted to be a direct target of hyperoside and was downregulated by hyperoside. Downregulation of RNF8 by hyperoside impeded the nuclear translocation of β-catenin and disrupted the Wnt/β-catenin pathway, which reduced the expression of the target genes c-myc, cyclin D1, and programmed death ligand 1 (PD-L1). Decreased PD-L1 levels contributed to induced immunity in Jurkat cells in vitro. Finally, in vivo studies demonstrated that hyperoside significantly reduced tumor size, inhibited PD-L1 and RNF8 expression, and induced apoptosis in tumor tissues of a subcutaneous mouse model. Hyperoside exerts its anti-PCa effect by reducing RNF8 protein, inhibiting nuclear translocation of β-catenin, and disrupting the Wnt/β-catenin pathway, in turn reducing the expression of PD-L1 and improving Jurkat cell immunity.

E3 ubiquitin ligase RNF180 impairs IPO4/SOX2 complex stability and inhibits SOX2-mediated malignancy in ovarian cancer.

RING finger protein 180 (RNF180), an E3 ubiquitin ligase, is thought to be a tumor suppressor gene. However, the detailed mechanism of its effect on ovarian cancer (OV) has not been elucidated. Importin 4 (IPO4) which belongs to transport protein is reported to have cancer-promoting effects on OV. Here, we explored the potential signaling pathways related to RNF180 and IPO4. It was first verified that RNF180 is downregulated and IPO4 is upregulated in OV. By overexpressing or knocking down RNF180 in OV cells, we confirmed that RNF180 inhibited the malignant behaviors of OV cells both in vitro and in vivo. Bioinformatics analysis and proteomics experiments found that RNF180 could interact with IPO4 and promote the degradation of IPO4 through ubiquitination. In addition, overexpression of IPO4 removed the inhibitory effect of RNF180 on OV. We subsequently found that IPO4 could bind to the oncogene Sex determining Region Y-box 2 (SOX2). Knockdown of IPO4 in OV cells decreased SOX2 protein level in nucleus and promoted cyclin-dependent kinase inhibitory protein-1 (p21) expression. Overexpression of RNF180 also inhibited the expression of SOX2 in nucleus. All these results indicated that RNF180 inhibited the nuclear translocation of SOX2 by promoting ubiquitination of IPO4, which ultimately promoted the expression of p21 and then suppressed the progression of OV. This study verified the tumor suppressor effect of RNF180 on OV, elucidated the mechanism of the molecule network related to RNF180 and IPO4 in OV and identified for OV.

Circ_0072088 promotes breast carcinoma progression via modulating miR-607/RNF2 axis.

To explore how the circular non-coding RNA circ_0072088 influences the progression of breast carcinoma (BC) by affecting cell behavior. We measured the levels of circ_0072088, microRNA-607 (miR-607), and ring finger protein 2 (RNF2) mRNA levels in BC tissues and cell lines using quantitative real-time PCR. We also conducted cell counting kit-8 (CCK-8), BrdU incorporation, and flow cytometry assays to assess cell viability, cell cycle, and apoptosis, respectively. We observed increased levels of circ_0072088 and RNF2, and decreased levels of miR-607 in BC tissues. Overexpressing circ_0072088 promoted BC cell proliferation and cell cycle progression while inhibiting apoptosis. Conversely, silencing circ_0072088 had the opposite effects. Our data suggest that circ_0072088 directly targets and downregulates miR-607, which in turn upregulates RNF2, a target of miR-607. Moreover, miR-607 overexpression could mitigate the pro-proliferative and anti-apoptotic effects of circ_0072088 on BC cells. Circ_0072088 drives BC progression by downregulating miR-607 and upregulating RNF2, thereby promoting cell proliferation and cycle progression while reducing apoptosis.

RIDDLE syndrome ring finger protein 168 deficiency in a 9-month-old boy from Jazan, Saudi Arabia

This study explores the intricate challenges of ring finger protein 168 (RNF168) deficiency, an uncommon genetic disorder characterized by immunodeficiency, radiation sensitivity, and diverse clinical traits. Presenting the case of a 9-month-old Saudi boy, our investigation emphasizes the critical importance of early diagnosis and intervention in managing this complex syndrome. Comparative analyses across global cases reveal substantial diversity in clinical features and genetic profiles, underscoring the need for ongoing research and international collaboration. Our findings significantly contribute to the understanding of RNF168 deficiency, guiding diagnostic and management strategies for improved patient outcomes. While acknowledging study limitations, particularly in comprehensive assessments, this research highlights the necessity for continued exploration to unravel the genetic and clinical intricacies of RNF168 deficiency. The insights gained from this study hold the potential for advancing knowledge and refining approaches to diagnose and manage this rare disorder effectively.

Non-adherent culture method affects the proliferation and apoptosis of mesenchymal stem cells through inhibiting LINC00707 to promote RNF6-mediated QKI ubiquitination

Exploration of the molecular mechanisms of mesenchymal stem cell (MSC) growth has significant clinical benefits. Long non-coding RNAs (lncRNAs) have been reported to play vital roles in the regulation of the osteogenic differentiation of MSCs. However, the mechanism by which lncRNA affects the proliferation and apoptosis of MSCs is unclear. In this study, sequencing analysis revealed that LINC00707 was significantly decreased in non-adherent human MSCs (non-AC-hMSCs) compared to adherent human MSCs. Moreover, LINC00707 overexpression promoted non-AChMSC proliferation, cell cycle progression from the G0/G1 phase to the S phase and inhibited apoptosis, whereas LINC00707 silencing had the opposite effect. Furthermore, LINC00707 interacted directly with the quaking (QKI) protein and enhanced the E3 ubiquitin-protein ligase ring finger protein 6 (RNF6)-mediated ubiquitination of the QKI protein. Additionally, the overexpression of QKI rescued the promotive effects on proliferation and inhibitory effects on apoptosis in non-AC-hMSCs induced by the ectopic expression of LINC00707. Thus, LINC00707 contributes to the proliferation and apoptosis in non-AChMSCs by regulating the ubiquitination and degradation of the QKI protein.

Ring Finger Protein 38 Induces Colorectal Cancer Proliferation by Activating the Phosphatidyl- Inositol 3-Kinase/Serine-Threonine Kinase Signaling

Previous studies have identified that ring finger protein 38 (RNF38) induces proliferative potential in many types of tumors. However, its functions in colorectal cancer (CRC) are unclear. This study aims to elucidate the regulatory effects of RNF38 on CRC proliferation in vitro and in vivo. Expression pattern and prognostic potential of RNF38 in clinically collected CRC cases were analyzed. After intervening RNF38 levels in SW480 and HT29 cells, viability, cell cycle progression and apoptosis were examined. Subsequently, we generated RNF38 overexpressed transgenic mice and xenograft model in nude mice. in vivo regulation of RNF38 on CRC proliferation was assessed. RNF38 was upregulated in CRC tissues. Overexpression of RNF38 stimulated proliferative ability and inhibited apoptosis in CRC cells. In addition, in vivo overexpression of RNF38 triggered tumor growth of CRC in nude mice. Silence of RNF38 markedly suppressed in vivo proliferation of CRC and induced apoptosis by activating the PI3K/AKT signaling.

M phase-specific interaction between SBDS and RNF2 at the mitotic spindles regulates mitotic progression

Shwachman-Diamond syndrome (SDS) is an autosomal recessive inherited disorder caused by biallelic mutations in the Shwachman-Bodian-Diamond syndrome (SBDS) gene. SBDS protein is involved in ribosome biogenesis; therefore SDS is classified as a ribosomopathy. SBDS is localized at mitotic spindles and stabilizes microtubules. Previously, we showed that SBDS interacts with ring finger protein 2 (RNF2) and is degraded through RNF2-dependent ubiquitination. In this study, we investigated when and where SBDS interacts with RNF2 and the effects of the interaction on cells. We found that SBDS co-localized with RNF2 on centrosomal microtubules in the mitotic phase (M phase), whereas SBDS and RNF2 localized to the nucleolus and nucleoplasm in the interphase, respectively. The microtubule-binding assay revealed that SBDS interacted directly with microtubules and RNF2 interacted with SBDS bound to microtubules. In addition, SBDS was ubiquitinated and degraded by RNF2 during the M phase. Moreover, RNF2 overexpression accelerated mitotic progression. These findings suggest that SBDS delays mitotic progression, and RNF2 releases cells from suppression through the ubiquitination and subsequent degradation of SBDS. The interaction between SBDS and RNF2 at mitotic spindles might be involved in mitotic progression as a novel regulatory cascade.

RNF6 promotes gastric cancer progression by regulating CCNA1/CREBBP transcription

ABSTRACT Ring finger protein 6 (RNF6) is a member of the E3 ubiquitin ligase family. Previous studies have reported the involvement of RNF6 as a ubiquitin ligase in the progression of gastric cancer (GC). However, this study found that RNF6 has a clear localization in the nucleus of GC, indicating a role other than ubiquitin ligase. Further chromatin immunoprecipitation sequencing (ChIP-seq) analysis revealed that RNF6 has DNA binding and transcriptional regulatory effects and is involved in important pathways such as tumor cell cycle and apoptosis. Cyclin A1 (CCNA1) and CREB binding protein (CREBBP) are downstream targets for RNF6 transcription regulation in GC. RNF6 binds to the promoter region of CCNA1/CREBBP and is actively regulating their expression in GC cells. Silencing CCNA1/CREBBP partially reversed the promoting effect of RNF6 overexpression on the biological function of GC cells. Our study suggests that RNF6 promotes the progression of GC by regulating CCNA1/CREBBP transcription.

Decabromodiphenyl ether induces the chromosome association disorders of spermatocytes and deformation failures of spermatids in mice

The environmental presence of decabromodiphenyl ether (BDE-209), which is toxic to the male reproductive system, is widespread. The current study investigated its mechanism of toxicity in mice. The results showed, that BDE-209 induced DNA damage, decreased the expression of the promoter of meiosis spermatogenesis- and oogenesis-specific basic helix-loop-helix 1 (Sohlh1), meiosis related-factors Lethal (3) malignant brain tumor like 2 (L3MBTL2), PIWI-like protein 2 (MILI), Cyclin-dependent kinase 2 (CDK2), Cyclin A, synaptonemal complex protein 1 (SYCP1) and synaptonemal complex protein 3 (SYCP3), and caused spermatogenic cell apoptosis, resulting in a decrease in sperm quantity and quality. Furthermore, BDE-209 downregulated the levels of anaphase-promoting complex/cyclosome (APC/C), increased the expression of PIWI-like protein 1 (MIWI) in the cytoplasm of elongating spermatids, and decreased the nuclear levels of RING finger protein 8 (RNF8), ubiquitinated (ub)-H2A/ub-H2B, and Protamine 1 (PRM1)/Protamine 2 (PRM2), while increasing H2A/H2B nuclear levels in spermatids. The reproductive toxicity was persistent for 50 days following the withdrawal of BDE-209 exposure. The results suggested that BDE-209 inhibits the initiation of meiosis by decreasing the expression of Sohlh1. Furthermore, the reduced expression of L3MBTL2 inhibited the formation of chromosomal synaptonemal complexes by depressing the expression of meiosis regulators affecting the meiotic progression and also inhibited histone ubiquitination preventing the replacement of histones by protamines, by preventing RNF8 from entering nuclei, which affected the evolution of spermatids into mature sperm.

The malignancy suppression and ferroptosis facilitation of BCL6 in gastric cancer mediated by FZD7 repression are strengthened by RNF180/RhoC pathway

BackgroundB-cell lymphoma 6 (BCL6) is a transcription repressor that plays a tumor suppressor or promoting role in various tumors. However, its function and molecular mechanism in gastric cancer (GC) remain unclear. Ferroptosis, a novel programmed cell death, is closely related to tumor development. In this research, we aimed to explore the role and mechanism of BCL6 in malignant progression and ferroptosis of gastric cancer.MethodsFirstly, BCL6 was identified as an important biomarker that attenuated the proliferation and metastasis of GC through tumor microarrays and confirmed in GC cell lines. RNA sequence was performed to explore the downstream genes of BCL6. The underlying mechanisms were further investigated by ChIP, dual luciferase reporter assays and rescue experiments. Cell death, lipid peroxidation, MDA and Fe2+ level were detected to determine the effect of BCL6 on ferroptosis and the mechanism was revealed. CHX, MG132 treatment and rescue experiments were used to explore the upstream regulatory mechanism of BCL6.ResultsHere we showed that BCL6 expression was significantly decreased in GC tissues, and patients with low BCL6 expression showed more malignant clinical features and poor prognosis. The upregulation of BCL6 may significantly inhibited the proliferation and metastasis of GC cells in vitro and in vivo. In addition, we found that BCL6 directly binds and transcriptionally represses Wnt receptor Frizzled 7 (FZD7) to inhibit the proliferation, metastasis of GC cells. We also found that BCL6 promoted lipid peroxidation, MDA and Fe2+ level to facilitate ferroptosis of GC cells by FZD7/β-catenin/TP63/GPX4 pathway. Furthermore, the expression and function of BCL6 in GC were regulated by the ring finger protein 180 (RNF180)/ras homolog gene family member C (RhoC) pathway, which had been elucidated to be involved in significantly mediating the proliferation and metastasis of GC cells.ConclusionsIn summary, BCL6 should be considered a potential intermediate tumor suppressor to inhibit the malignant progression and induce ferroptosis, which might be a promising molecular biomarker for further mechanistic investigation of GC.

PD-L1 expression downregulation by RNF43 in gastric carcinoma enhances antitumour activity of T cells.

Ring finger protein 43 (RNF43), a transmembrane E3 ubiquitin ligase, has been indicated to be a potential biomarker for gastric cancer treatment, as this protein increases tumour cell apoptosis and suppresses cellular proliferation. The role of RNF43 in cellular immunotherapy remains unclear. Herein, we aimed to explore the expression level of RNF43 in gastric cancer cell lines and its role in cellular immunotherapy. The expression level of RNF43 and PD-L1 and their correlation in gastric cancer cell lines were analysed. The expression of PD-L1 was negatively correlated with that of RNF43 in gastric cancer cell lines. RNF43 interacted with PD-L1 to augment both K48- and K63-linked ubiquitination of PD-L1 in gastric cancer cell lines. In addition, RNF43 expression in gastric cancer cell lines could enhance the antitumour activity of T cells. In conclusion, this study reveals that RNF43 can inhibit PD-L1 expression to enhance the antitumour activity of cellular immunotherapy.

Abstract 3504: Evaluating the molecular determinants of PDAC racial health disparities

Abstract Pancreatic cancer is the 3rd leading cause of cancer-related deaths with a 5-year survival rate of just 11.5%. Pancreatic ductal adenocarcinoma (PDAC), accounting for over 90% of pancreatic malignancies, has the highest incidence rate, mortality rate, and shortest survival times in non-Hispanic black (defined here as African and/or African American ancestry) patients compared to other races. Due to the racial health disparities of PDAC, this study seeks to quantify the proteomic signatures associated with tumor racial origin to identify the underlying molecular pathways that could contribute to these disparities. Using mass spectrometry on primary PDAC tumor samples collected from 30 Caucasian and 12 African American patients, 183 proteins were differentially expressed between these groups. The most over-expressed protein in African American tumors was Ring Finger Protein 2 (RNF2) with a log2 fold-change greater than six. RNF2 had previously been found to regulate GATA Binding Protein 6 (GATA6) in embryonic stem cells. Additionally, GATA6 is also an important determinant in PDAC subtyping with higher GATA6 expression leading to greater chemosensitivity. To expand upon these findings, chromatin immunoprecipitation and sequencing (ChIP-seq) was performed on MIA-PaCa2 to identify binding sites for RNF2 in PDAC cells. Peaks were called with MACS2 (q <0.01) and a final peak list with an irreproducible discovery rate (IDR) of less than 0.05 across two replicates was generated. From the 1,142 replicated peaks identified, RNF2 peaks were localized over the transcription start site of 644 unique transcripts, including GATA6. Modulating RNF2 levels by exogenous expression or short hairpin RNA (shRNA) knockdown confirms that GATA6 is a target of RNF2 repression in PDAC. To further identify its downstream targets in PDAC, RNF2 knock down in MIA-PaCa2 was analyzed by RNA-seq. Differential gene expression analysis revealed RNF2 knockdown decreased expression of genes related to innate immunity, which we have previously identified as a marker of the aggressive Inflammatory PDAC subtype using proteomics. Of the 4,763 differentially expressed genes (P-adj < 0.05), 226 had RNF2 binding sites, demonstrating the scope of RNF2 regulation in MIA-PaCa2. Our work identifies differences in the underlying proteome of PDAC tumors as a function of racial origin, with overexpression of RNF2 in African American tumors potentially contributing to inflammation and subtype delineation. Because PDAC subtypes are predictive of therapeutic response, additional research is necessary to determine whether targeting RNF2 can mitigate oncogenic phenotypes that contribute to racial health disparities. Citation Format: Anthony E. Johansen-Sallee, Alexa M. Barber, Henry Law, Jose Trevino, Nicholas T. Woods. Evaluating the molecular determinants of PDAC racial health disparities [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 3504.

Abstract 6134: Exploiting the oncogenic state of colorectal cancer for therapeutic development

Abstract Over the past two decades, only incremental progresses have been made in the therapeutic management of colorectal cancer (CRC). This is in part attributed to the fact that the Wnt pathway, the oncogenic pathway that initiates the transformation of CRC and sustains tumor maintenance, has been largely undruggable. While most efforts targeting Wnt signaling have revolved around interfering with Wnt target genes that regulate cell growth, we turned our attention to a subset of genes encoding for negative regulators of the pathway. Of particular interest are the cell-surface E3 ubiquitin ligases, ring finger protein 43 (RNF43) and ZNRF3, two known negative regulators of Wnt signaling that promote the turnover of Wnt receptors Frizzled/low-density lipoprotein receptor-related proteins (FZD/LRPs) via ubiquitination-mediated degradation. Given their exposed extracellular domains (ECDs), we set out to explore whether this intrinsic cellular degradative machinery could be repurposed for an antibody-based targeted protein degradation platform. More specifically, we have devised a technology that exploits the elevated expression of ZNRF3 and RNF43 in colon cancer by generating bispecific antibodies that can tether these E3 ubiquitin ligases to a variety of cell surface receptors to promote tumor receptor degradation. Most notably, these antibody degraders are highly active in vivo and can degrade receptors in a tumor specific manner while sparing normal tissues. This platform, that we have been dubbed Proteolysis Targeting Antibodies (PROTABs) describes a strategy for the rapid development of potent, bioavailable and tissue-selective degraders of cell-surface proteins. Citation Format: Felipe de Sousa e Melo. Exploiting the oncogenic state of colorectal cancer for therapeutic development. [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 6134.

RNF6 activates TGF-β1/c-Myb pathway to promote EMT in esophageal squamous cell carcinoma.

This study aimed to investigate RING-Finger Protein 6 (RNF6) expression in esophageal squamous cell carcinoma (ESCC) cells and whether it affects cell proliferation, invasion, and migration by regulating the TGF-β1/c-Myb pathway. TCGA database was used to analyze RNF6 expression in normal tissues and esophageal cancer tissues. Kaplan-Meier method was used to examine the correlation between RNF6 expression and patient prognosis. SiRNA interference vector and RNF6 overexpression plasmid were constructed, and RNF6 was transfected into Eca-109 and KYSE-150 esophageal cancer cell line. In vitro scratch assay and Transwell assay were conducted to investigate the effects of RNF6 on the migration and invasion of Eca-109 and KYSE-150 cells. RT-PCR detected the expression of Snail, E-cadherin, and N-cadherin, and TUNEL detected the apoptosis of cells. RNF6 up-regulation promoted the progression of esophageal cancer and predicted poor prognosis. RNF6 also enhanced the migration and invasion of ESCC cells in vitro. RNF6 silencing inhibited the migration and invasion of ESCC cells. TGF-β inhibitors reversed the oncogenic effects of RNF6. RNF6 regulated the migration and invasion of ESCC cells by activating the TGF-β pathway. RNF6/TGF-β1 promoted esophageal cancer progression through c-Myb. RNF6 promotes the proliferation, invasion, and migration of ESCC cells possibly by activating the TGF-β1/c-Myb pathway and affects the progression of ESCC.

Emerging Roles of RNF168 in Tumor Progression.

RING finger protein 168 (RNF168) is an E3 ubiquitin ligase with the RING finger domain. It is an important protein contributing to the DNA double-strand damage repair pathway. Recent studies have found that RNF168 is significantly implicated in the occurrence and development of various cancers. Additionally, RNF168 contributes to the drug resistance of tumor cells by enhancing their DNA repair ability or regulating the degradation of target proteins. This paper summarizes and prospects the research progress of the structure and main functions of RNF168, especially its roles and the underlying mechanisms in tumorigenesis.

A comparative assessment of RNF38 and P53 genes expression in the sperm samples obtained from males with normozoospermia and asthenospermia: A case-control study.

Infertility is considered as a common problem appears in about 10-12% of couples in their reproductive ages. Ring finger protein 38 (RNF38) gene is a ubiquitin-protein ligase that can regulate Protein 53 (P53) and affect cellular motility. Considering the role of P53 on cellular motility and RNF38 on the regulation of P53, the present study aimed to assess the difference between RNF38 and P53 genes expression in normozoospermic and asthenospermic samples as a diagnostic biomarker in males. The present study was conducted among 21 asthenospermicsand 63 healthy individuals. First, the real-time polymerase chain reaction technique was applied to measure the expression level of the P53 and RNF38 genes extracted from sperm samples, and the glyceraldehyde-3phosphate dehydrogenase gene was selected as the reference gene. An increase and a decrease occurred in the level of P53 and RNF38 genes expressions in asthenospermic and normozoospermic samples, respectively. In addition, a significant difference was observed between increasing P53 gene expression (p 0.001), reducing RNF38 one, and decreasing sperm motility (p 0.001) in asthenospermic cells compared to that of normozoospermic ones. Based on the results, an increase in the expression of the P53 gene and a decrease in the expression of the RNF38 gene had a significant relationship with asthenospermia in men. Therefore, it is expected that an effective step should be adopted to diagnose the asthenospermia expression pattern by using these results.