CC Cells Research Articles

Enhanced Adsorption Kinetics and Capacity of a Stable CeF3@Ni3N Heterostructure for Methanol Electro‐Reforming Coupled with Hydrogen Production

Alkaline methanol‐water electrolysis system is regarded as an appealing strategy for electro‐reforming methanol into formate and producing hydrogen with low energy‐consumption compared with alkaline water electrolysis. However, stability and selectivity under high current densities for practical application remain challenging. Herein, a CeF3@Ni3N nanosheets array anchored on carbon cloth (CeF3@Ni3N/CC) was fabricated. The gradual extrusion of F species from Ni(OH)2 lattices can stabilize hierarchical structure and construct abundant heterostructure interfaces. Moreover, CeF3 can modulate electron distribution of Ni3N, thus simultaneously enhancing the surface adsorption kinetics and capability of methanol and OH‐, which is conducive to enhanced methanol oxidation reaction (MOR) activity and selectivity. Therefore, bifunctional CeF3@Ni3N/CC exhibits low potential of 1.43 V at 500 mA cm‐2, along with high stability over 72 h and high faradaic efficiency (FEs) in MOR, as well as an overpotential of 76 mV to achieve 50 mA cm‐2 for hydrogen evolution reaction (HER). Furthermore, membrane‐free CeF3@Ni3N/CC||CeF3@Ni3N/CC cell for MOR||HER delivers high electrocatalytic activity, long‐term stability and FEs at high current density of 300 mA cm‐2. This study highlights the importance of optimizing surface adsorption behavior of active species, as well as rational design of highly efficient heterostructure electrocatalysts for methanol upgrading coupled with hydrogen production.

Enhanced Adsorption Kinetics and Capacity of a Stable CeF3@Ni3N Heterostructure for Methanol Electro-Reforming Coupled with Hydrogen Production.

Alkaline methanol-water electrolysis system is regarded as an appealing strategy for electro-reforming methanol into formate and producing hydrogen with low energy-consumption compared with alkaline water electrolysis. However, stability and selectivity under high current densities for practical application remain challenging. Herein, a CeF3@Ni3N nanosheets array anchored on carbon cloth (CeF3@Ni3N/CC) was fabricated. The gradual extrusion of F species from Ni(OH)2 lattices can stabilize hierarchical structure and construct abundant heterostructure interfaces. Moreover, CeF3 can modulate electron distribution of Ni3N, thus simultaneously enhancing the surface adsorption kinetics and capability of methanol and OH-, which is conducive to enhanced methanol oxidation reaction (MOR) activity and selectivity. Therefore, bifunctional CeF3@Ni3N/CC exhibits low potential of 1.43 V at 500 mA cm-2, along with high stability over 72 h and high faradaic efficiency (FEs) in MOR, as well as an overpotential of 76 mV to achieve 50 mA cm-2 for hydrogen evolution reaction (HER). Furthermore, membrane-free CeF3@Ni3N/CC||CeF3@Ni3N/CC cell for MOR||HER delivers high electrocatalytic activity, long-term stability and FEs at high current density of 300 mA cm-2. This study highlights the importance of optimizing surface adsorption behavior of active species, as well as rational design of highly efficient heterostructure electrocatalysts for methanol upgrading coupled with hydrogen production.

Metabolic Response to Small Molecule Therapy in Colorectal Cancer Tracked with Raman Spectroscopy and Metabolomics.

Despite numerous screening tools for colorectal cancer (CRC), 25 % of patients are diagnosed with advanced disease. Novel diagnostic technologies that are early, accurate, and rapid are imperative to assess the therapeutic efficacy of clinical drugs and identify new biomarkers of treatment response. Here Raman spectroscopy (RS) was used to track metabolic reprogramming in KRAS-mutant HCT116 and SW837 cells, and KRAS wild-type CC cells. RS combined with multivariate analysis methods distinguished nonresponsive, partially responsive, and responsive cells treated with cetuximab, a monoclonal antibody for EGFR inhibition, sotorasib, a clinically approved KRAS inhibitor, and various doses of trametinib, an inhibitor of the MAPK pathway. Cells treated with a combination of subtoxic doses of trametinib and BKM120, an inhibitor of the PI3K pathway, showed a synergistic response between the two pathways. Using a supervised machine learning regression model, we established a scoring methodology trained to a priori predict therapeutic response to new treatment combinations. RS metabolites were verified with mass spectrometry, and enrichment pathways were identified, including amino acid, purine, and nicotinate and nicotinamide metabolism that differentiated monotherapy from combination therapy. Our approach may ultimately be applicable to patient-derived primary cells and cultures of patient tumors to predict effective drugs for individualized care.

PARP1 acetylation at K119 is essential in regulating the progression and proliferation of cervical cancer cells.

Cervical cancer, CC, is one of the malignant cancers in women worldwide. Many studies about the genesis and progression of CC have been done at genomic, transcriptional, translational, and epigenetic levels. However, much less is done at post-translational modification (PTM) level. We first used pan-PTM antibodies to compare the pan PTM levels between clinical normal cervical tissues and CC tissues; we then sent the selected samples for label-free identification of acetylation sites. Next, we employed WT or K119A mutant PARP1-EGFP-STREPII plasmid transfection in Hela cells and examined various indexes including colony formation, wound healing, ROS generation, early apoptosis, and immunofluorescence and quantification of proliferation markers (Ki67, PCNA, and p-P53). Last, we examined the levels of multiple important kinases regulating cervical cancer progression. We found that pan-acetylation was the most downregulated in clinical CC samples, whereas the acetylation of PARP1, Poly(ADP-ribose) polymerase-1, was upregulated at K119. Next, we showed that PARP1-WT overexpression significantly suppressed the proliferation and progression in CC cell line Hela, while K119A overexpression didn't show any impact. Finally, PARP1-WT overexpression significantly decreased p-ERK1/2 while didn't affect the phosphorylation levels of other important kinases such as AKT, MTOR, and RPS6. This study discovered a new type of PTM of PARP1 in CC, and showed that PARP1 acetylation at K119 is essential in regulating the proliferation and progression of CC through ERK1/2. Further studies are required to investigate how PARP1 acetylation impact its function.

Study on the Commercial Metalized Plastic Current Collector PET-Cu and PP-Cu Toward High-Energy Lithium-Ion Battery.

Commercial metalized plastic current collector (MPCC) is receiving widespread attention from the business and academic communities, due to its properties of excellent electrical conductivity and low mass density. However, the application of MPCC on the side of copper is rarely studied. Herein, sandwich-like polyethylene terephthalate-based (PET) and polypropylene-based (PP) copper (Cu) current collectors via magnetron sputtering and electroplating are fabricated. Most importantly, the electrical performance, mechanical safety quality, and revealed the corresponding failure mechanism for the MPCC cells are first systematically evaluated. First, during the 45°C electrical cycling tests, PET-Cu CC (82.67%) and PP-Cu CC (82.32%) cells both have comparable capacity retention with the traditional Cu CC (Tra-Cu CC) cell (84.55%) after 500 cycles. The slight reduction in the cycling performance is induced by the crack of the Cu layer around the embedded SiO2 particle for PET-Cu CC cell and the detachment of Cu layer for PP-Cu CC cell. Second, during the nail-penetration test, MPCC cells maintain no fire and explosion for more than 5min, since the heat-shrinkable function of polymeric film can interrupt the continuous Joule heat released by internal short-circuit. This work provides important guidance for the large-scale application of MPCC in the field of lithium-ion batteries.

Metabolic Response to Small Molecule Therapy in Colorectal Cancer Tracked with Raman Spectroscopy and Metabolomics

AbstractDespite numerous screening tools for colorectal cancer (CRC), 25 % of patients are diagnosed with advanced disease. Novel diagnostic technologies that are early, accurate, and rapid are imperative to assess the therapeutic efficacy of clinical drugs and identify new biomarkers of treatment response. Here Raman spectroscopy (RS) was used to track metabolic reprogramming in KRAS‐mutant HCT116 and SW837 cells, and KRAS wild‐type CC cells. RS combined with multivariate analysis methods distinguished nonresponsive, partially responsive, and responsive cells treated with cetuximab, a monoclonal antibody for EGFR inhibition, sotorasib, a clinically approved KRAS inhibitor, and various doses of trametinib, an inhibitor of the MAPK pathway. Cells treated with a combination of subtoxic doses of trametinib and BKM120, an inhibitor of the PI3K pathway, showed a synergistic response between the two pathways. Using a supervised machine learning regression model, we established a scoring methodology trained to a priori predict therapeutic response to new treatment combinations. RS metabolites were verified with mass spectrometry, and enrichment pathways were identified, including amino acid, purine, and nicotinate and nicotinamide metabolism that differentiated monotherapy from combination therapy. Our approach may ultimately be applicable to patient‐derived primary cells and cultures of patient tumors to predict effective drugs for individualized care.

Mediation of circ_0007142 on miR-128-3p/S100A14 pathway to stimulate the progression of cervical cancer.

A previous study has confirmed the upregulation of circ_0007142 expression in CC. Here, we aimed to investigate the effect and mechanism of circ_0007142 in CC progression. The expression of circ_0007142, microRNA-128-3p (miR-128-3p), S100 calcium-binding protein A14 (S100A14), and epithelial mesenchymal transition (EMT)-related markers was measured by qRT-PCR and Western blot. Cell proliferative, migratory, and invasion abilities were evaluated using cell counting Kit-8, cell colony formation, 5-ethynyl-2'-deoxyuridine, and transwell assays, respectively. The interaction among circ_0007142, miR-128-3p and S100A14 was identified by dual-luciferase reporter and RNA immunoprecipitation assays. In vivo experiment was implemented to investigate the effect of circ_0007142 on tumor growth. CC tissues and cells displayed high expression of circ_0007142 and S100A14, and low expression of miR-128-3p in comparison to the controls. Knockdown of circ_0007142 resulted in the inhibition of cell proliferation, migration invasion, and EMT in vitro. In support, circ_0007142 deficiency hindered tumor growth and EMT in vivo. In rescue experiments, downregulation of miR-128-3p relieved circ_0007142 absence-mediated anticancer impacts. MiR-128-3p overexpression-induced inhibitory effects on cell growth and metastasis were attenuated by S100A14 overexpression. Importantly, circ_0007142 regulated S100A14 expression by sponging miR-128-3p. Circ_0007142 knockdown suppressed CC cell malignant behaviors by miR-128-3p/S100A14 pathway, providing a possible circRNA-targeted therapy for CC.

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.

Abstract 5664: Genetic alterations and pharmacological response profiles from 10 cervical cancer cell lines

Abstract Background: There are significant parallels between CC cell lines (CCCL) and newly diagnosed tumors, the most prominent being HPV infection/integrations and PI3K as well as epigenetic pathway alterations. Objectives: Identify genomic alterations associated with resistance to specific drug families in pharmacological profiles in Cell Lines (CL) and in Primary Tumors. Material and Methods: 16 public and 4 newly patient-derived CL have been extensively characterized by HPV type, genetic (WES) and protein (RPPA) profiles. Pharmacological profiling against 32 drugs was carried out in triplicate samples and repeated on 10 selected CL. Variant calling of small-scale alterations (SNVs, InDels) was performed using GATK HaplotypeCaller (v4.0.2.1). Variants annotation was performed with Annovar (v 2017/07/06) using respectively gene (refGene), known polymorphims (avsnp v147, 1000G v2015/08, ESP6500v2, ExAC) and cancer databases (COSMIC v70, ICGC v21) as well as deleterious impact predictors (dbNSFP v33). Copy number alterations were assessed using Facets (v0.5.11) with 4 blood samples from the RAIDs tumor project used to generate a “baseline” copy number profiles for CLs. Results: All 10 cell lines had strikingly similar DSS (drug sensitivity scores) to a family of four different microtubule targeting agents (MTAs). Bioinformatics subtraction of genetic alterations present in resistant (CRL 10302, CRL 7920, CRL 1550) and absent in sensitive CLs: (IC1, IC3, IC5), revealed a consistent LoF (loss of function) alteration in one single gene: PABPC3 ‘Poly A tail binding protein, cytoplasmic C3’, a gene involved in messenger RNA stability and translational initiation. Additionally, 50 gene alterations from the pathway “POST TRANSLATIONAL MODIFICATIONS OF TUBULIN” were variably detected in resistant CLs. In primary tumors from the RAIDS database, alterations in PABPC3 were present in 10/301 (3%), half of which documented by at least one database: (Cosmic, ICGC, Cancer Hotspot). All CLs were sensitive to Bortezomib, Omipalisib and Palbociclib. A large spectrum activity to Vorinostat, (HDAC inhibitor) in line with frequent (sensitizing) LoF alterations in epigenetic suppressor genes, contrasted with a selective response to other well-known CC drugs: Methotrexate, Gemcitabine, Sorafenib. Three out of CL were highly sensitive to APR246 (Eprenetapopt) as single agent. Bioinformatics gene subtraction identified OSBPL1A, a marker of synthetic lethality in the ferroptosis pathway, in all three CLs. Discussion and Conclusion: A string of LoF alterations (CSMD2/CSMD3, ZNF717, CDC27) had been previously associated with poor response to radiation or to chemo-radiation, in tumors and are present in most CLs which are derived from resistant tumors. Future objectives are to validate the present biomarker findings in the context of a clinical platform trial. Citation Format: Suzy Scholl, Pierre Gestraud, Elodie Girard, Lolita Lecompte, Maral Halladjian, Leanne de Koning, Nicolas Servant, Maud Kamal, Elaine Del Nery. Genetic alterations and pharmacological response profiles from 10 cervical cancer cell lines [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 5664.

Ochratoxin A promotes chronic enteritis and early colorectal cancer progression by targeting Rinck signaling.

Mycotoxins, such as aflatoxin and ochratoxin A (OTA), are found at measurable levels in many staple foods; the health implications of long-term exposure of such toxins are poorly understood. Increasing evidence has confirmed the important role of OTA in upregulation of oxidative stress- and inflammatory response-induced tissue injury. However, it remains unknown whether ochratoxin A can promote chronic colitis and its associated colon cancer (CRC) development, and potential molecular mechanism. Additionally, RING finger-interacting protein with C kinase (RINCK) is a ubiquitin ligase and mediates immune response. Unfortunately, the potential molecular function of RINCK on regulation of colitis is still largely unknown. This study aims to provide mechanistic evidence that the role of RINCK in colitis and early colorectal cancer progression in response to OTA treatment via targeting nuclear factor erythroid 2-related factor 2 (NRF2). The Cancer Genome Atlas (TCGA) database, GEO database, human subjects with CC phenotype and CC cell lines were used in this work. Pathological links between OTA, RINCK and treatment of CC are revealed through comprehensive means such as biological information analysis, clinical experiments, RNA-seq, and verification experiments. In this study, under oxidative stress in setting of colitis, we first identified RINCK as a key regulatory factor and a novel endogenous suppressor of nuclear factor erythroid 2-related factor 2 (NRF2), and we also confirm that RINCK is a NRF2 partner protein that catalyses its ubiquitination and degradation in intestinal epithelial cells (IECs). Notably, in vivo study, pathological phenotypes triggered by OTA pretreatment, accompanied by post-treatment of dextran sulfate sodium (DSS)-induced colitis was significantly mitigated by IEC-specific deficiency of Rinck, IEC-Rinck(KO) and adenovirus-associated virus (AAV)-triggered suppression of Rinck in rodent model, and lentivirus (LV)-mediated downregulation of Rinck (LV-shRinck) in rabbit model, as determined by decreased endogenous reactive oxygen species (ROS) production, pro-inflammatory cytokines contents, improved body weights, reduced survival rates, restored colon length, assuasive DAI and histological scores. Inversely, transgenic mice by IEC-specific Rinck overexpression, IEC-Rinck(OE) accelerated colitis in acute or chronic colitis rodent models and in vitro experiments. Moreover, we found that OTA pretreatment-promoted azoxymethane (AOM)/DSS-induced colitis-associated early colorectal cancer (CRC) was also dramatically reduced by IEC-Rinck(KO), indicated by the decreased tumor number and corresponding KI-67 levels. Clinical samples analysis revealed that RINCK levels were greatly increased in tumor tissues of patients with CRC phenotypes. In parallel, RINCK deletion remarkably retarded the proliferation of colon cancer and tumor growth in vitro and in vivo, respectively. Mechanistically, in response to onset of colitis, RINCK directly interacts with NRF2 and promotes ubiquitin-proteasome degradation via increasing K48-linkage ubiquitin chain, thus leads in suppression of NRF2 nuclear translocation and its downstream cascade inactivation, which retards antioxidant defense. The findings suggested that oral sub-chronic exposure of OTA significantly facilitates DSS-induced colitis and colitis-associated CRC development. These results further elucidated the potential role of RINCK in colitis progression by mediating NRF2 degradation, and could be considered as a therapeutic target for the treatment of such disease.

Circular RNA circ_0000119 promotes cervical cancer cell growth and migration via miR-433-3p/PAK2 axis.

The purpose of this study was to investigate the role of circ_0000119 on CC progression and its molecular mechanism. The expression levels of circ_0000119, miR-433-3p, and p21-activated kinase 2 (PAK2) in CC tissues and cell lines were measured by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was assessed using 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay, 5-Ethynyl-2'-deoxyuridine (EdU) assay and colony formation assay. Cell cycle and apoptosis were assessed by flow cytometry. Cell migration and invasive ability were examined by Transwell assays. Downstream binding targets of circ_0000119 were predicted by online bioinformatics tools and confirmed by dual luciferase reporter gene assay, RNA immunoprecipitation (RIP) assay, and RNA pull-down assay. The role of circ_0000119/miR-433-3p/PAK2 axis in regulating the CC process was explored by rescue experiments. A xenograft model was constructed to further determine the effect of circ_0000119 on CC tumor growth in vivo. Immunohistochemistry (IHC) assay was conducted for Ki67 expression. Circ_0000119 was aberrantly upregulated in CC tissues and cell lines. Knockdown of circ_0000119 inhibited CC cell proliferation, cell cycle progress, migration, invasion, and promoted apoptosis of CC cells. MiR-433-3p was a binding target of circ_0000119, and PAK2 was a downstream gene of miR-433-3p. MiR-433-3p inhibition reversed the inhibitory effect of silencing circ_0000119 on CC progression. In addition, PAK2 overexpression reversed the effect of miR-433-3p on CC progression. PAK2 expression was regulated by circ_0000119 and miR-433-3p. Moreover, circ_0000119 knockdown reduced tumor growth of CC in vivo. Circ_0000119 was upregulated in CC, and circ_0000119 knockdown suppressed CC malignant development through the miR-433-3p/PAK2 axis.

A triple-drug combination induces apoptosis in cervical cancer-derived cell lines.

Cervical cancer is a worldwide health problem due to the number of deaths caused by this neoplasm. In particular, in 2020, 30,000 deaths of this type of tumor were reported in Latin America. Treatments used to manage patients diagnosed in the early stages have excellent results as measured by different clinical outcomes. Existing first-line treatments are not enough to avoid cancer recurrence, progression, or metastasis in locally advanced and advanced stages. Therefore, there is a need to continue with the proposal of new therapies. Drug repositioning is a strategy to explore known medicines as treatments for other diseases. In this scenario, drugs used in other pathologies that have antitumor activity, such as metformin and sodium oxamate, are analyzed. In this research, we combined the drugs metformin and sodium oxamate with doxorubicin (named triple therapy or TT) based on their mechanism of action and previous investigation of our group against three CC cell lines. Through flow cytometry, Western blot, and protein microarray experiments, we found TT-induced apoptosis on HeLa, CaSki, and SiHa through the caspase 3 intrinsic pathway, including the critical proapoptotic proteins BAD, BAX, cytochrome-C, and p21. In addition, mTOR and S6K phosphorylated proteins were inhibited in the three cell lines. Also, we show an anti-migratory activity of the TT, suggesting other targets of the drug combination in the late CC stages. These results, together with our former studies, conclude that TT inhibits the mTOR pathway leading to cell death by apoptosis. Our work provides new evidence of TT against cervical cancer as a promising antineoplastic therapy.

The BMP inhibitor follistatin-like 1 (FSTL1) suppresses cervical carcinogenesis.

Follistatin-like 1 (FSTL1) is a cancer-related matricellular secretory protein with contradictory organ-specific roles. Its contribution to the pathogenesis of cervical carcinoma is still not clear. Meanwhile, it is necessary to identify novel candidate genes to understand cervical carcinoma's pathogenesis further and find potential therapeutic targets. We collected cervical carcinoma samples and matched adjacent tissues from patients with the locally-advanced disease and used cervical carcinoma cell lines HeLa and C33A to evaluate the effects of FSTL1 on CC cells. The mRNA transcription and protein expression of FSTL1 in cervical carcinoma tumor biopsy tissues were lower than those of matched adjacent tissues. Patients with a lower ratio of FSTL1 mRNA between the tumor and its matched adjacent tissues showed a correlation with the advanced cervical carcinoma FIGO stages. High expression of FSTL1 markedly inhibited the proliferation, motility, and invasion of HeLa and C33A. Regarding mechanism, FSTL1 plays its role by negatively regulating the BMP4/Smad1/5/9 signaling. Our study has demonstrated the tumor suppressor effect of FSTL1, and these findings suggested a potential therapeutic target and biomarker for cervical carcinoma.

Silencing of RhoC induces macrophage M1 polarization to inhibit migration and invasion in colon cancer via regulating the PTEN/FOXO1 pathway.

Ras homologue family member C (RhoC) is an oncogene in diverse types of human cancers, whereas its regulatory mechanisms involving macrophage polarization is rarely investigated. This study is designed to explore the regulatory role of RhoC in colon cancer and the underlying molecular mechanisms involving macrophage polarization. We detected RhoC expression by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot, and analysed the biological function of RhoC knockdown in CC cells by the MTT, wound healing and transwell assay. Macrophage polarization-associated markers, genes associated with migration, phosphatase and tensin homologue (PTEN) and forkhead box O (FOXO) were determined by qRT-PCR and western blot. The xenograft tumour mouse model was used to assess the role of RhoC in vivo. RhoC is highly expressed in CC cells. The cell viability, invasion and migration abilities of CC cells were reduced by knockdown of RhoC. RhoC knockdown promoted M1 polarization, inhibited M2 polarization and decreased levels of genes associated with migration (matrix metalloproteinase-2 and matrix metalloproteinase-9). Silencing of RhoC inhibited tumour growth and expression of genes associated with migration in the xenografted model. In addition, silencing of RhoC promoted PTEN/FOXO1 expression, and PTEN inhibitor (SF1670) reversed the inhibitory effects of RhoC silencing. We demonstrated that silencing of RhoC reduced CC cells invasion and migration, and tumour growth by suppressing M2 macrophage polarization via regulating the PTEN/FOXO1 pathway.

Abstract PR010: Inhibition of HGF maturation overcomes cetuximab resistance in colorectal cancer

Abstract We have previously identified that activation of receptor tyrosine kinases (RTKs), MET and RON contributed to resistance to the EGF receptor (EGFR)-directed therapeutic antibody, cetuximab. These findings originated from our in vitro 3D type I collagen cultures of human colorectal cancer (CRC) cell line HCA-7 derivatives CC, SC, and CC-CR. CC are sensitive to cetuximab, while SC and CC-CR are resistant. Both de novo and acquired modes of cetuximab resistance in SC and CC-CR, respectively, could be overcome by crizotinib, a multi-RTK inhibitor that also targets MET and RON. Conversely, exogenous administration of MET ligand, HGF could transiently induce cetuximab resistance which could be further overcome by crizotinib addition. HGF/HGFL are synthesized as inactive precursors and require cleavage by proteases (HGFA, Matriptase, and Hepsin) to be biologically active. To inhibit HGF/HGFL cleavage in cetuximab-resistant cells, we employed inhibitors of HGF/HGFL proteases (ZFH7116 and VD2173) and were able to overcome both de novo and acquired cetuximab resistance. A survey of TCGA datasets indicated that HGF/HGFL were overexpressed in several CRC CMS subtypes. We next expressed human HGF in cetuximab-sensitive CC cells and observed that HGF overexpression imparts cetuximab resistance. Moreover, cetuximab resistance induced by HGF overexpression could be overcome by the downstream MET inhibition (with crizotinib), and we are now testing if the upstream inhibition of HGF proteases (with ZFH7116/VD2173) also overcomes cetuximab resistance. Combined these results indicate that inhibition of HGF cleavage and maturation may be a novel way to overcome resistance to EGFR-targeted therapies in CRC. Citation Format: Ramona Graves-Deal, Vivian T. Jones, Galina T. Bogatcheva, Zheng Cao, Sarah J. Harmych, Vishnu Damalanka, Lidija Klampfer, Robert J. Coffey, James W. Janetka, Bhuminder Singh. Inhibition of HGF maturation overcomes cetuximab resistance in colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr PR010.

CircRNA hsa_circ_0001627 aggravates cervical cancer progression through upregulation of FNDC3B and activating PI3K/mTOR signaling pathway.

Circular RNAs (CircRNAs) are key regulators in the development and progression of human cancers. However, the biological roles and mechanisms of circRNAs in gastric cancer (GC) remain largely unknown. Analyzing circRNA microarray dataset (GSE102686) and clinical specimens, a novel circRNA termed hsa_circ_0001627, was identified and it was highly expressed in CC cancerous tissues and cells, and was associated with poor clinical outcomes. Functionally, hsa_circ_0001627 silencing impaired the malignant progression of CC cells and the growth of CC xenografts in nude mice. Mechanistically, hsa_circ_0001627 acted as a miR-1225-5p sponge, thus indirectly regulating FNDC3B and leading to the activation of PI3K/mTOR signaling pathway. Collectively, the present study indicates that hsa_circ_0001627 regulates miR-1225-5p/FNDC3B/PI3K/mTOR axis and functions as an oncogene in CC progression, suggesting the potential therapeutic use of hsa_circ_0001627 in CC treatment.

Circ_0002762 Accelerates Glycolysis Metabolism to Promote Cervical Cancer Progression via the miR-526b-5p/HK2 Axis

Objectives: The purpose of this study was to investigate the role and mechanism of circ_0002762 in CC. Design: This study was designed for silencing circ_0002762 in CC cells and xenograft tumor models to investigate the role of circ_0002762 in CC in vitro and in vivo. Materials and Methods: The relative expression levels of circ_0002762, miR-526b-5p, and hexokinase2 (HK2) in CC tissues and cells were detected by real-time quantitative polymerase chain reaction or Western blot. Glycolysis-related extracellular acidification rate, glucose production, lactic acid consumption, and ATP levels were measured using the appropriate kits. Cell proliferation was assessed by 5-ethynyl-2′-deoxyuridine and colony formation assay. Cell apoptosis was detected by flow cytometry. The binding relationship between miR-526b-5p and circ_0002762 or HK2 was verified by dual-luciferase reporter assay and RNA pull-down assay. Tumor growth in vivo was detected by xenograft tumor model. Results: The expressions of circ_0002762 and HK2 were up-regulated and miR-526b-5p was down-regulated in CC tissues and cells. Circ_0002762 knockdown inhibited glycolysis and proliferation and promoted apoptosis of CC cells. In addition, miR-526b-5p suppression reversed the inhibition of CC development induced by circ_0002762 silencing. HK2 overexpression eliminated the inhibition of miR-526b-5p on CC progression. Moreover, silencing of circ_0002762 inhibited CC tumor growth in vivo. Limitations: The practical application of circ_0002762 in clinical practice needs further investigation. Conclusion: Circ_0002762 knockdown inhibited CC progression by regulating miR-526b-5p/HK2 axis, suggesting that circ_0002762 was a promising therapeutic strategy for CC.

Abstract 5240: S-phase cyclinA associated protein in the ER(SCAPER)rs3812908 T>C promotes the nuclear translocation of CyclinA2 and enhances DNA damage repair in radioresistance of colorectal cancer

Abstract Radiotherapy resistance is still an obstacle of improving the survival of patients with locally advanced colorectal cancer. In this study, whole-exome sequencing and targeted sequencing were preformed on colorectal cancer tumor tissue samples which were colloected from multiple centers, and the pathologic complete response predictive model PGS-LARC(Prediction Genotype Signature for Locally Advanced Rectal Cancer) was established. A candidate SNV-SCAPER rs3812908 T>C was screened using this model. To investigate the relationship between SCAPER rs3812908 T>C and radiosensitivity of colorectal cancer, targeted sequencing was performed and the results revealed that proportion of CC genotype was significantly higher in resistant sequencing cohort than in sensitive ones (30% vs. 22%). And Immunohistochemistry (IHC) results revealed that SCAPER protein expressed less in CC genotype colorectal cancer tissues than in TT genotype. Here, the cell proliferation results showed that SCAPER-KD cells and SCAPER rs3812908 CC cells were more radioresistant than SCAPER-NC and TT cells. Furthermore, the immunofluorescence assay showed SCAPER rs3812908 T>C promoted translocation of S phase associated protein-Cyclin A2 into nucleus and western blotting results showed that the phosphorylation of ERK and JNK(p-ERK and p-JNK) increased significantly in SCAPER CC cells, and the protein p-ATR, p-ATM, p-CDC25C and CDC25A related to homologous recombination repair were also increased in SCAPER-KD and SCAPER CC cells.Taken together, the SCAPER gene rs3812908 T>C plays an important role in radioresistance of colorectal cancer. The SNV of SCAPER rs3812908 T>C significantly promotes Cyclin A2 nuclear translocation, activates the MAPKs pathway, and increases homologous recombination repair, and finally leads to radioresistance. Citation Format: Hai-na Yu, Ting Jiang, Shuang Liu, Yan Yuan, Shao-yan Xi, Zhi-wei Guo, Yuan-hong Yuan, Yan Li, Wei-wei Xiao. S-phase cyclinA associated protein in the ER(SCAPER)rs3812908 T>C promotes the nuclear translocation of CyclinA2 and enhances DNA damage repair in radioresistance of colorectal cancer [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 5240.

Abstract 1525: Inhibition of HGF maturation overcomes cetuximab resistance in colorectal cancer

Abstract We have previously identified that activation of receptor tyrosine kinases (RTKs) MET and RON contributed to resistance to the EGF receptor (EGFR)-directed therapeutic antibody cetuximab. These findings originated from our in vitro 3D type I collagen cultures of human colorectal cancer (CRC) cell line HCA-7 derivatives CC, SC, and CC-CR. CC are sensitive to cetuximab, while SC and CC-CR are resistant. Both de novo and acquired modes of cetuximab resistance in SC and CC-CR, respectively, could be overcome by crizotinib, a multi-RTK inhibitor that also targets MET and RON. Conversely, exogenous administration of MET ligand, HGF could transiently induce cetuximab resistance which could be further overcome by crizotinib addition. HGF/HGFL are synthesized as inactive precursors and require cleavage by proteases (HGFA, Matriptase, and Hepsin) to be biologically active. To inhibit HGF/HGFL cleavage in cetuximab-resistant cells, we employed inhibitors of HGF/HGFL proteases (ZFH7116 and VD2173) and were able to overcome both de novo and acquired cetuximab resistance. A survey of TCGA datasets indicated that HGF/HGFL were overexpressed in several CRC CMS subtypes. We next expressed human HGF in cetuximab-sensitive CC cells and observed that HGF overexpression imparts cetuximab resistance. Moreover, cetuximab resistance induced by HGF overexpression could be overcome by the downstream MET inhibition (with crizotinib) and we are now testing if the upstream inhibition of HGF proteases (with ZFH7116/VD2173) also overcomes cetuximab resistance. Combined these results indicate that inhibition of HGF cleavage and maturation may be a novel way to overcome resistance to EGFR-targeted therapies in CRC. Citation Format: Ramona Graves-Deal, Galina Bogatcheva, Vivian T. Truong, Vishnu Damalanka, Lidija Klampfer, Robert J. Coffey, James W. Janetka, Bhuminder Singh. Inhibition of HGF maturation overcomes cetuximab resistance in colorectal cancer [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 1525.

The E6 Oncoprotein of HPV16 AA-c Variant Regulates Cell Migration through the MINCR/miR-28-5p/RAP1B Axis.

The E6 oncoprotein of HPV16 variants differentially alters the transcription of the genes involved in migration and non-coding RNAs such as lncRNAs. The role of the lncRNA MINCR in cervical cancer and its relationship with variants of oncogenic HPV remain unknown. Therefore, the objective of this study was to analyze the effect of the E6 oncoprotein of the AA-c variant of HPV16 in cell migration through the MINCR/miR-28-5p/RAP1B axis. To explore the functional role of MINCR in CC, we used an in vitro model of C33-A cells with exogenous expression of the E6 oncoprotein of the AA-c variant of HPV16. Interfering RNAs performed MINCR silencing, and the expression of miR-28-5p and RAP1B mRNA was analyzed by RT-qPCR. We found that C33-A/AA-c cells expressed MINCR 8-fold higher compared to the control cells. There is an inverse correlation between the expression of miR-28-5p and RAP1B in C33-A/AA-c cells. Our results suggest that MINCR might regulate the expression of RAP1B through the inhibition of miR-28-5p in CC cells expressing the E6 oncoprotein of HPV16 AA-c. We report, for the first time, that the MINCR/miR-28-5p/RAP1B axis positively regulates cell migration in CC-derived cells that express the E6 oncoprotein of the AA-c variant of HPV16.