Malignant Cell Phenotype Research Articles

Abstract PR018: Inflammation-induced mechanotransduction is necessary and sufficient to create pre-cancerous squamous lung metaplasias and necessary to drive progression to dysplasia

Abstract Chronic exposure to tobacco smoke triggers chronic inflammation, fostering a fibrotic microenvironment and promoting the focal replacement of columnar human bronchial epithelial cells (hBECs) with cells exhibiting squamous morphology, termed metaplasia. How squamous metaplasia progresses to squamous dysplasia and, ultimately, squamous lung carcinoma is under intense investigation. Using human bronchial tissues, we demonstrate that, both in vitro and in vivo, cellular stress associated with chronic inflammation (e.g., oxidative stress, DNA damage) enhances TGF-β signaling and creates a fibroblast state exhibiting upregulated HSP47 expression, driving increased collagen fiber alignment, elevated tissue stiffness, and subsequent activation of YAP-dependent mechanotransduction in adjacent hBECs. These Stress/Tension-producing Instructive Fibroblasts (STIFs) alone were sufficient to induce hBECs to squamous metaplasia, precursors to tumor formation. Furthermore, when tumor suppressor function was compromised in hBECs, STIFs induced dysplastic phenotypes. Mechanistically, STIFs act in a dominant manner modulating epithelial cell identity and malignant phenotypes via extracellular matrix (ECM)-dependent mechanotransduction. Inhibition of HSP47-dependent collagen processing effectively prevented fibroblast-induced squamous metaplasia and dysplasia and was capable of reversing fibroblast-induced metaplasia, restoring bronchial epithelial cell identity. Citation Format: Thea D. Tisty, Deng Pan, Philippe Gascard, Joseph Caruso, Lorenzo Ferri, Chira Chen-Tanyolac. Inflammation-induced mechanotransduction is necessary and sufficient to create pre-cancerous squamous lung metaplasias and necessary to drive progression to dysplasia [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr PR018.

LINC01614 Promotes Oral Squamous Cell Carcinoma by Regulating FOXC1

Background: Long non-coding RNAs (lncRNAs) are pivotal mediators during the development of carcinomas; however, it remains to be investigated whether lncRNAs are implicated in oral squamous cell carcinoma (OSCC). Methods: In this study, quantitative real-time PCR was conducted for detecting the expression of LINC01614 in OSCC cell lines. The biological functions of LINC01614 were assessed by loss- and gain-of-function experiments conducted both in vivo and in vitro. Cellular proliferation, migration, and invasion were investigated herein, and dual luciferase reporter assays were additionally performed to explore the relationships among LINC01614, miR-138-5p, and Forkhead box C1 (FOXC1). Results: The research presented herein revealed that OSCC cells express high levels of LINC01614. Functional experiments employing cellular and animal models demonstrated that LINC01614 knockdown repressed the malignant phenotypes of OSCC cells, including their growth, invasiveness, and migration. Further investigation revealed that LINC01614 absorbs miR-138-5p miRNA by functioning as a competing endogenous RNA to downregulate the abundance of FOXC1. Conclusions: The findings revealed that LINC01614 contributes to the progression of OSCC by targeting the FOXC1 signaling pathway. The study provides insights into a novel mechanistic process to regulate the development of OSCC, and established a possible target for the therapeutic management of OSCC.

SSX2IP promotes cell proliferation and migration in breast cancer by regulating FANCI.

Synovial sarcoma X breakpoint 2 interacting protein (SSX2IP) is expressed in various normal tissues and participates in the progression of human cancers. Nevertheless, the specific functions and underlying molecular mechanisms of SSX2IP in cancer, particularly in breast cancer, remain poorly understood. In this study, we aimed to explore the functional role of SSX2IP in breast cancer. Immunohistochemical staining, quantitative real-time PCR, and western blotting blot analysis were used to assess genes expression levels. By manipulating SSX2IP expression levels and conducting functional assays including Celigo cell counting assay or CCKCCK-8-8 assay, flow cytometry, wound healing assay, and Transwell assay, we explored the impact of SSX2IP on the malignant phenotype of breast cancer cells. Additionally, the in vivo tumor-suppressive ability of SSX2IP was investigated by tumor xenograft experiment. Our results revealed an upregulation of SSX2IP in the breast cancer. Functional assays demonstrated that SSX2IP knockdown inhibited cell proliferation and migration, induced apoptosis in vitro, as well as suppressed the tumor growth in vivo. Conversely, SSX2IP overexpression contributed to the malignant phenotype of breast cancer cells. Co-expression analysis showed that FA Complementation Group I (FANCI) was co-expressed with SSX2IP. Additionally, SSX2IP positively regulated FANCI expression and its interaction was verified by Co-IP.Co-IP. Furthermore, FANCI overexpression partially reversed the effects of SSX2IP knockdown on cell proliferation and metastasis. In summary, our findings revealed that SSX2IP contributes to the progression of breast cancer by regulating FANCI, hinting at its potential as a novel biomarker and therapeutic target for the treatment of breast cancer.

VCP enhances autophagy-related osteosarcoma progression by recruiting USP2 to inhibit ubiquitination and degradation of FASN

Osteosarcoma (OS) is a highly aggressive malignant tumor with a high rate of disability and mortality rates, and dysregulated autophagy is a crucial factor in cancer. However, the molecular mechanisms that regulate autophagy in OS remain unclear. This study aimed to explore key molecules that affect autophagy in OS and their regulatory mechanisms. We found that fatty acid synthase (FASN) was significantly increased in activated autophagy models of OS and promoted OS proliferation in an autophagy-dependent manner, as detected by LC3 double-labeled fluorescence confocal microscopy, western blotting, transmission electron microscopy (TEM), and cell functional experiments. Furthermore, co-immunoprecipitation combined with mass spectrometry (Co-IP/MS), ubiquitination modification, molecular docking, and protein truncation methods were used to identify FASN-interacting proteins and analyze their effects on OS. Valosin-containing protein (VCP) enhanced the FASN stability by recruiting ubiquitin specific peptidase-2 (USP2) to remove the K48-linked ubiquitin chains from FASN; domain 2 of VCP and the amino acid sequence () of USP2 were critical for their interactions. Gain- and loss-of-function experiments showed that the inhibition of FASN or USP2 attenuated the stimulatory effect of VCP overexpression on autophagy and the malignant phenotypes of OS cells in vitro and in vivo. Notably, micro-CT indicated that VCP induced severe bone destruction in nude mice, which was abrogated by FASN or USP2 downregulation. In summary, VCP recruits USP2 to stabilize FASN by deubiquitylation, thereby activating autophagy and promoting OS progression. The identification of the VCP/USP2/FASN axis, which mediates autophagy regulation, provides important insights into the underlying mechanisms of OS and offers potential diagnostic and therapeutic strategies for patients with OS.

Molecular and functional profiling of primary normal ovarian cells defines insights into cancer development and drug-responses

Patients with ovarian cancer, especially the high-grade serous (HGSOC) subtype, face poor prognosis due to late diagnosis and treatment resistance. Owing to the high heterogeneity of HGSOC, identifying the origin of the disease and optimal treatments is difficult. Here, we characterized two primary immortalized human ovarian cell lines, HOSE1C and HOSE2C, comparing their molecular profiling with representative HGSOC cells. We identified molecular features associated with normal and malignant phenotype of ovarian cells by applying single-cell transcriptomics, high-content imaged-based Cell Painting, and high-throughput drug testing. Our findings reveal distinct transcriptomic and morphological profiles for the two HOSEs, with a stromal phenotype. Moreover, their responses to the tumor microenvironment differ, exemplified by STAT1 and GREM1 upregulation in HOSE1C and HOSE2C, respectively. We identified selective activation of ERK/MEK targeted inhibitors in cancer cells compared to HOSEs. This study offers insights into the normal and malignant ovarian cells, shedding light on cancer development and drug responses.

Mini and enhanced CRISPR activators for cancer therapies

IntroductionThe RNA-guided nuclease Cas9 can be used as a programmable transcription activator, but there is still room for improvement in its effectiveness in eukaryotes, and its potential in cancer genetic therapy has been poorly investigated. ObjectivesWe aim to construct optimized CRISPRa tools and detect their potential role in cancer therapy by screening 9aa-TAD. MethodsWe selected a range of transcriptional coactivators for programmable activation and analyzed their effects on the expression of multiple endogenous genes using Flow cytometry and qRT-PCR. In order to improve the activation capacity of the CRISPRa tool, we fused the coactivators with the efficient dCas9-VPR system to construct a new activation system. Utilize RNA-seq to assess the activation specificity of genome-wide. To evaluate the value of the newly constructed activation system in cancer gene therapy, we activated the expression of the tumor suppressor genes PER2 and ZNF382, and performed changes in cancer cell proliferation qRT-PCR and clonal formation analysis. ResultsIn this study, we screened the NHR module from C. elegans, which demonstrated a high transcription activation capacity with a compact size compared to VP64. We successfully demonstrated its efficiency in activating endogenous genes in mammalian cells. Furthermore, we developed an enhanced fused variant called NHR-VP64-p65-Rta (NVPR), which showed even higher efficiency compared to the previously established VPR module, making it an effective CRISPRa tool. The dCas9-NVPR complex also exhibited high specificity on a genome-wide scale. Finally, we utilized the dCas9-NVPR tool to restore the expression of tumor suppressor genes PER2 and ZNF382, effectively inhibiting the malignant phenotype of cancer cells. ConclusionWe have successfully developed and demonstrated a breakthrough CRISPRa tool with promising implications for cancer genetic therapy. This innovation expands the range of available gene editing tools and further validates the immense potential of CRISPR-based approaches in precision medicine.

Identification of Cellular Senescence-Related Critical Genes and Molecular Classification and Revealing the Drug-Resistant Therapeutic Effect of IGFBP2 in Chronic Myeloid Leukemia.

The occurrence and development of hematologic tumors are closely linked to cellular senescence. However, the molecular characteristics associated with this phenomenon in chronic myeloid leukemia (CML) have not been thoroughly investigated. The cellular senescence score was calculated using gene set variation analysis. Consensus clustering algorithm was used to identify the molecular subtypes associated with cellular senescence. Clinical samples were collected for sequencing analysis to verify the expression of critical cellular senescence-related genes (CSRG). The effect of targeted inhibition of IGFBP2 on the malignant phenotype of CML-resistant cells was studied by cell experiments. The cellular senescence score in CML samples was significantly lower compared to normal samples. Higher expression of immune checkpoint markers correlated with increased cellular senescence scores. We identified two distinct molecular subtypes (C1 and C2) related to cellular senescence. The C1 subtype exhibited enhanced metabolic function and DNA damage repair capacity, while the C2 subtype showed higher infiltration of immune effector cells and activity in immune-related signaling pathways. We also discovered a group of drugs that displayed significant sensitivity differences between these two molecular subtypes, with the C2 subtype showing greater responsiveness to immunotherapy. Four critical cellular senescence-related genes (CSRGs), namely IGFBP2, IL7R, PLAU, and SUN1 demonstrated high diagnostic value for CML. We validated the expression levels of these four genes using clinical samples and confirmed through cell experiments that targeted inhibition of IGFBP2 effectively suppressed proliferation of resistant CML cells, promoted apoptosis, and enhanced therapeutic sensitivity to imatinib. Our study conducted a comprehensive analysis on CSRG expression characteristics in CML and explored potential correlations between cellular senescence and immune function. The identification of molecular subtypes provides valuable insights into assessing individual patients' biological characteristics for guiding clinical treatment decisions. Additionally, IGFBP2has emerged as a promising therapeutic target for therapy-resistant cases of CML.

RNA methylase RBM15 facilitates malignant progression of colorectal cancer through regulating E2F2 in an m6A modification-dependent manner.

Recently, RBM15 has emerged as an oncogenic factor in a majority of tumors. However, the mechanism is unclear that accounts for how RBM15-induces colorectal cancer (CRC) progression and it is in need of further study. We determined RBM15 expression through the UALCAN database and RT-qPCR. The role of RBM15 in inducing the malignant and aggressive cancerous phenotype was characterized based on the results of the western blot, RT-qPCR, CCK-8 and transwell assays. The target genes of RBM15 were screened by LinkedOmics. m6A methylation kit was applied to analyze the methylation levels of mRNA. SRAMP website was employed to predict m6A sites of targeted mRNA. RIP, dual luciferase reporter gene and actinomycin D assay were conducted to verify the interactions between RBM15 and its targeted gene, and the presence of m6A modification site of its targeted mRNA, respectively. We confirmed the augmentation of RBM15 expression in CRC, which also has a high clinical diagnostic value for CRC. Functionally, RBM15 silencing clearly restrained malignant cellular processes in CRC cells. Mechanistically, RBM15 bound to E2F2 which increased its m6A binding and stabilized the corresponding E2F2 mRNA formation. Excessive E2F2 largely restored the repression malignant phenotype of tumor cells caused by RBM15 silencing. RBM15 regulated E2F2 in an m6A modification-dependent manner thereby boosting malignant cellular processes in CRC. The RBM15/E2F2 axis may be a novel target for CRC therapy.

Immunosuppressive SOX9-AS1 Resists Triple-Negative Breast Cancer Senescence Via Regulating Wnt Signalling Pathway.

Long noncoding RNAs (lncRNAs) are involved in the regulation of triple-negative breast cancer (TNBC) senescence, while pro-carcinogenic lncRNAs resist senescence onset leading to the failure of therapy-induced senescence (TIS) strategy, urgently identifying the key senescence-related lncRNAs (SRlncRNAs). We mined seven SRlncRNAs (SOX9-AS1, LINC01152, AC005152.3, RP11-161 M6.2, RP5-968 J1.1, RP11-351 J23.1 and RP11-666A20.3) by bioinformatics, of which SOX9-AS1 was reported to be pro-carcinogenic. Invitro experiments revealed the highest expression of SOX9-AS1 in MDA-MD-231 cells. SOX9-AS1 knockdown inhibited cell growth (proliferation, cycle and apoptosis) and malignant phenotypes (migration and invasion), while SOX9-AS1 overexpression rescued these effects. Additionally, SOX9-AS1 knockdown facilitated tamoxifen-induced cellular senescence and the transcription of senescence-associated secretory phenotype (SASP) factors (IL-1α, IL-1β, IL-6 and IL-8) mechanistically by resisting senescence-induced Wnt signal (GSK-3β/β-catenin) activation. Immune infiltration analysis revealed that low SOX9-AS1 expression was accompanied by a high infiltration of naïve B cells, CD8+ T cells and γδ T cells. In conclusion, SOX9-AS1 resists TNBC senescence via regulating the Wnt signalling pathway and inhibits immune infiltration. Targeted inhibition of SOX9-AS1 enhances SASP and thus mobilises immune infiltration to adjunct TIS strategy.

Berberine inhibits the malignant cell phenotype by inactivating PI3K/AKT/mTOR signaling in laryngeal squamous cell carcinoma.

Berberine is an active compound found in different herbs used in Chinese medicine and is well-known for its potential anticancer properties. The study aimed to figure out the role of berberine in regulating the malignant behavior of laryngeal squamous cell carcinoma (LSCC) cells. LSCC cell lines (SNU-899 and AMC-HN-8) were treated with different concentrations of berberine (0-200 μM) to determine its cytotoxicity. The migration, invasion, and apoptosis of LSCC cells were measured by wound healing assays, Transwell assays, and flow cytometry. Western blot was performed for the quantification of proteins involved in PI3K/AKT/mTOR signaling. The viability of LSCC cells was dose-dependently reduced by berberine. Berberine dampened LSCC cell migration and invasion while augmenting cell apoptosis, as evidenced by a reduced wound closure rate, a decrease in invaded cell number, and a surge in cell apoptosis in the context of berberine stimulation. Importantly, the effects of berberine on the cancer cell process were enhanced by LY294002 (an inhibitor for PI3K) treatment. Moreover, the protein levels of phosphorylated PI3K, AKT, and mTOR were markedly reduced in response to berberine treatment. Berberine inhibits cell viability, migration, and invasion but augments cell apoptosis by inactivating PI3K/AKT/mTOR signaling in LSCC.

CKIP-1 inhibits M2 macrophage polarization to suppress the progression of gastric cancer by inactivating JAK/STAT3 signaling.

Gastric cancer (GC) is a frequently occurring malignancy with poor prognosis. Casein kinase 2 interacting protein-1 (CKIP-1) is a PH domain-containing protein implicated in regulating tumorigenesis and macrophage homeostasis. This study aimed to elucidate the role and potential mechanism of CKIP-1 in the progression of GC. CKIP-1 expression in GC tumor and para-carcinoma tissues was detected using RT-qPCR. Then, human monocyte cell line THP-1 was treated with PMA, interleukin (IL)-4 and IL-13 to induce M2-polarized macrophages. CD206, arginase-1 (Arg-1) and transforming growth factorβ1 (TGFβ1) expression in M2-polarized macrophages with or without CKIP-1 overexpression was evaluated. Moreover, GC cell lines (MKN45 and HGC27 cells) were co-cultured with CKIP-1-overexpressed M2-polarized macrophages, and the viability, migration and invasion of GC cells were measured. Additionally, immunoblotting assessed the expression of JAK/STAT3 signaling-related proteins and STAT3 agonist Colivelin was used to treat GC cells to perform the rescue experiments to analyze the changes of malignant phenotypes of GC cells. Results showed that CKIP-1 was downregulated in GC tissues and M2-polarized macrophages. CKIP-1 overexpression inhibited M2 macrophage polarization and decreased TGFβ1 secretion. Besides, elevated CKIP-1 expression in M2-polarized macrophages inhibited the viability, migration and invasion of GC cells. Furthermore, CKIP-1 overexpression inactivated JAK2/STAT3 signaling in GC cells by inhibiting TGFβ1 level. Specifically, Colivelin treatment abrogated the influences of CKIP-1 upregulation on the malignant phenotypes of GC cells. Collectively, CKIP-1 inhibits M2 macrophage polarization to suppress the progression of GC by inactivating JAK/STAT3 signaling pathway.

UBE2C promotes LUAD progression by ubiquitin-dependent degradation of p53 to inactivate the p53/p21 signaling pathway

Lung adenocarcinoma (LUAD) is one of the greatest causes of cancer death worldwide. As a novel potential tumor biomarker, ubiquitin-conjugating enzyme E2C (UBE2C) is a critical factor during the onset and development of human cancers. However, the mechanisms of UBE2C in LUAD are not well understood. In this study, increased expression level of UBE2C was observed in LUAD tumor tissues. High LUAD level portended a worse prognosis of LUAD patients. Down-regulation of UBE2C attenuated the cell proliferation and cycle, migration, and invasion. Consistently, the tumorigenic capacity of LUAD cells in nude mice was significantly suppressed by the knockdown of UBE2C. Knockdown of UBE2C inhibited the degradation of p53 protein via an ubiquitin-proteasome pathway, thereby increasing p53 and p21 protein expression. Moreover, the inhibition of LUAD cell malignant phenotypes caused by UBE2C knockdown was attenuated on account of the inactivation of p53/p21 signaling pathway. In conclusion, UBE2C facilitates cell malignant behaviour in LUAD by ubiquitin-dependent degradation of p53 to suppress the p53/p21 signaling pathway. UBE2C is potentially developed as a therapeutic target for patients with LUAD.

Colorectal fibroblasts promote malignant phenotype of colorectal cancer cells by activating the ERK signaling pathway

To investigate the effect of human colorectal fibroblast (CCD-18Co)-conditioned medium (CCD18-Co-CM) on biological behaviors of colorectal cancer (CRC) cells and explore the possible molecular mechanisms. Real-time cellular analysis (RTCA), clone formation assay and wound healing assay were used to analyze the changes in proliferation, clone formation, and migration abilities of CRC cell lines HCT116 and Caco-2 treated with CCD18-Co-CM. Western blotting was used to detect the changes in ATK, ERK and STAT3 signaling pathways in the CRC cells activated by CCD18-Co-CM. The effect of CCD18-Co-CM on spheroidization ability of the cells was assessed with sphere-formation assay, and the changes in expressions of CRC stemness markers were detected using RT-PCR. CCD-18Co-CM significantly promoted proliferation, colony formation, and migration of HCT116 and Caco-2 cells, enhanced sphere-forming ability and expressions of CRC stemness markers, and increased ERK phosphorylation in the cells. Treatment with SCH772984 effectively inhibited CCD-18Co-CM-induced ERK signaling pathway activation, suppressed the malignant phenotype, and lowered the sphere-forming ability and expression of stemness markers of the two CRC cells. Colorectal fibroblasts promote malignant phenotype of CRC cells by activating the ERK signaling pathway.

Prostate cancer cell-derived exosomes ZNF667-AS1 reduces TGFBR1 mRNA stability to inhibit Treg expansion and DTX resistance by binding to U2AF1

BackgroundDocetaxel (DTX) resistance attenuates anti-tumor effects of DTX on prostate cancer (mCRPC) and drug resistance was related to Treg expansion in tumors. ZNF667-AS1 played a suppressing role in various tumors and tumor-derived exosomes carry lncRNAs to participate in tumor progression. Here, the effects of ZNF667-AS1 on malignant characteristics and DTX resistance in PC and the effect and its underlying molecular mechanism of tumor-derived exosomes carrying ZNF667-AS1 on Treg expansion were investigated.MethodsThe identification of exosomes were determined using TEM, NTA and western blot. The abundance of genes and proteins were evaluated using IHC, RT-qPCR, western blot and FISH. Malignant phenotypes of PC cells were evaluated by means of Edu, scratch test, transwell, CCK-8 and flow cytometry. The percentage of CD4+CD25+Foxp3+ Tregs was detected using flow cytometry. The location of ZNF667-AS1 was detected using nuclear-cytoplasmic fractionation. The co-location of ZNF667-AS1 and U2AF1 protein was detected using IF-FISH assay. The interactions among ZNF667-AS1, TGFBR1 and U2AF1 were verified using RNA pull-down, RIP and dual luciferase activity.ResultsZNF667-AS1 expression in PC samples was lowered, which was negatively relative to poor prognosis and DTX resistance. ZNF667-AS1 overexpression inhibited malignant phenotypes of PC cells, tumor growth and DTX resistance. Besides, DTX resistant cell-derived exosomes expressed lower ZNF667-AS1 expression. Exosomes carrying exogenously high ZNF667-AS1 expression derived PC cells or serum of mice suppressed Treg expansion. On the mechanism, ZNF667-AS1 interacted with U2AF1 to destabilize TGFBR1 mRNA and reduce TGFBR1 expression in CD4+T cells.ConclusionZNF667-AS1 suppressed cell growth of PC cells, tumor growth of mice and DTX resistance to PC cells and exogenously high ZNF667-AS1 expression in tumor-derived exosomes destabilized TGFBR1 mRNA and reduce TGFBR1 expression through interacting with U2AF1, thus resulting in attenuated Treg expansion, which was related to DTX resistance.

METTL3 facilitates the progression of cervical cancer by m6A modification-mediated up-regulation of NEK2.

N6-methyladenosine (m6A) is the most prevalent modification found in eukaryotic RNA and played a significant role in various cancers. However, the mechanism by which m6A modification influences cervical cancer (CC) tumorigenesis remains unclear. Therefore, we aim to elucidate the role and mechanism of METTL3 in CC progression. In the present study, we observed a significant upregulation of METTL3 in CC tissues and cell lines. Knockdown of METTL3 resulted in reduced growth, migration, and invasion of CC cells, as well as affected apoptosis, while overexpression of METTL3 reversed these effects. Through a combined analysis of meRIP-seq and Ribo-seq data following METTL3 knockdown, NEK2 was identified as a key target of METTL3 in CC cells. Correlation analysis, MeRIP-qPCR, and luciferase reporter assay suggested that METTL3 regulates NEK2 expression through m6A modification. NEK2 synergized with METTL3 to mediate the malignant phenotype of CC cells. The METTL3-NEK2 axis promoted CC progression by activating the Wnt/β-catenin pathway and inhibiting the apoptosis pathway. In conclusion, METTL3 facilitated the malignant progression of CC and contributed to the formation of the METTL3-NEK2 regulatory axis in an m6A-dependent manner, which represented a potential target for CC therapy.

ADAMTS4 exacerbates lung cancer progression via regulating c-Myc protein stability and activating MAPK signaling pathway

BackgroundLung cancer is one of the most frequent cancers and the leading cause of cancer-related deaths worldwide with poor prognosis. A disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) is crucial in the regulation of the extracellular matrix (ECM), impacting its formation, homeostasis and remodeling, and has been linked to cancer progression. However, the specific involvement of ADAMTS4 in the development of lung cancer remains unclear.MethodsADAMTS4 expression was identified in human lung cancer samples by immunohistochemical (IHC) staining and the correlation of ADAMTS4 with clinical outcome was determined. The functional impact of ADAMTS4 on malignant phenotypes of lung cancer cells was explored both in vitro and in vivo. The mechanisms underlying ADAMTS4-mediated lung cancer progression were explored by ubiquitination-related assays.ResultsOur study revealed a significant upregulation of ADAMTS4 at the protein level in lung cancer tissues compared to para-carcinoma normal tissues. High ADAMTS4 expression inversely correlated with the prognosis of lung cancer patients. Knockdown of ADAMTS4 inhibited the proliferation and migration of lung cancer cells, as well as the tubule formation of HUVECs, while ADAMTS4 overexpression exerted opposite effects. Mechanistically, we found that ADAMTS4 stabilized c-Myc by inhibiting its ubiquitination, thereby promoting the in vitro and in vivo growth of lung cancer cells and inducing HUVECs tubule formation in lung cancer. In addition, our results suggested that ADAMTS4 overexpression activated MAPK signaling pathway.ConclusionsWe highlighted the promoting role of ADAMTS4 in lung cancer progression and proposed ADAMTS4 as a promising therapeutic target for lung cancer patients.

SPAG4 enhances mitochondrial respiration and aerobic glycolysis in colorectal cancer cells by activating the PI3K/Akt signaling pathway.

Aerobic glycolysis plays a pivotal role in the progression of tumors. Previously, a glycolysis-associated prognostic model in CRC was constructed and the glycolysis-related gene SPAG4 was discovered to be upregulated in CRC and was correlated with adverse prognosis. To date, however, no study has elucidated the specific role of SPAG4 in the development of CRC. In our study, CRC cells were transfected with si-SPAG4 or OE-SPAG4 to evaluate the influence of SPAG4 silencing or overexpression on CRC cell malignant behaviors. CRC cell proliferation and metastasis were detected via CCK-8, colony formation, and Transwell assays. The oxygen consumption rate and extracellular acidification rate of CRC cells were determined by using an XF24 extracellular flux analyzer. The expression of SPAG4, key mitochondrial markers (NDUFA1, SDHB, ATP5A, and PGC-1α), key enzymes involved in glycolysis (GLUT1, HK2, LDHA, PKM2, and PFK1), and PI3K/Akt pathway-molecules and downstream transcription factor HIF-1α was assessed by RT-qPCR and western blot analysis. SPAG4 expression in CRC and normal tissue samples was tested through immunohistochemical staining. Finally, SPAG4-overexpressed CRC cells were treated with LY294002 to validate the inhibition of PI3K/Akt pathway on CRC cell malignant phenotypes. Our results showed that SPAG4 was upregulated in CRC cells and tissues, and high expression SPAG4 predicted shorter overall survival time. SPAG4 knockdown inhibited while SPAG4 overexpression enhanced CRC cell proliferation, migration, invasion, mitochondrial respiration, and aerobic glycolysis. Overexpressing SPAG4 elevated p-PI3K, p-Akt, p-mTOR, and HIF-1α protein levels, which were restored after LY294002 treatment. Furthermore, LY294002 abolished the promotion of SPAG4 overexpression on CRC malignant phenotypes. Collectively, SPAG4 plays an oncogenic role in CRC by promoting mitochondrial respiration and aerobic glycolysis through activating the PI3K/Akt signaling. These findings suggest that inhibition of SPAG4-mediated glucose metabolism may represent a potential strategy for the clinical treatment of CRC.

Circ_0008035 Promotes Gastric Cancer Development via the miR-429/SMAD2 Cascade.

The vital roles of circular RNAs (circRNAs) in human tumorigenesis have attracted more attention. Circ_0008035 is one of the most up-regulated circRNAs in gastric cancer (GC). Herein, we explored the associated mechanism of circ_0008035 in GC. EdU incorporation experiments were performed to monitor cell proliferation ability. Cell cycle progression, apoptosis, angiogenesis, migration, and invasion were analyzed using flow cytometry, Tube formation, and Transwell assays respectively. Protein expression was detected by Western blot. Dual-luciferase reporter experiments were applied to demonstrate the relationship between circ_0008035 or SMAD family member 2 (SMAD2) and microRNA-429 (miR-429). Mouse xenograft assays were conducted for evaluation of the role of circ_0008035 in vivo. Circ_0008035 content was elevated in GC tissues (P < .0001) and cell lines (P < .001), and its deficiency hindered GC cell proliferation (P < .01), HUVEC angiogenesis (P < .05), and GC cell metastasis (P < .01) and triggered apoptosis (P < .01). Circ_0008035 could sponge miR-429 to up-regulate SMAD2 expression (P < .0001). Circ_0008035 absence restrained tumor growth in vivo (P < .01). MiR429 was a mediator of circ_0008035 function, and miR-429 hindered GC cell malignant phenotypes by SMAD2. Circ_0008035 aggravates GC cell malignant progression partially by targeting the miR-429/SMAD2 axis. Considering the inhibitory effect of circ_0008035 deficiency on GC progression, targeting circ_0008035 may be a potential approach to prevent or treat GC.

FTX promotes esophageal cancer progression and desensitizes esophageal cancer cells to ionizing radiation by microRNA-99a/b-3p/WEE1/ERCC1 axis

Abstract Objectives This study was conducted to uncover the underlying cellular and molecular mechanisms of FTX dysregulation in EC. Methods The gene expression in tumor tissues was detected using western blot, immunohistochemistry, and quantitative real-time polymerase chain reaction (qPCR). The dual-luciferase reporter and RNA FISH assays confirmed the interaction between miRNA and target genes. Mouse models for Xenograft and lung metastasis were used to assess EC cell tumorigenesis and metastasis. Results This study finds that up-regulated FTX in patients with esophageal cancer correlates with poor clinical outcomes. Silencing FTX inhibits esophageal cancer cell growth and migration in vitro and tumor metastasis in vivo. miR-99a/b-3p sensitizes esophageal cancer cells to ionizing radiation by WEE1 (Wee1-like protein kinase) and ERCC1 (excision repair cross-complementation group 1) in vitro. Conclusions FTX promotes the malignant biological phenotype of esophageal cancer cells. Mechanistically, FTX acts as a ceRNA to regulate the transcription of WEE1 and ERCC1 by sponging hsa-miR-99a/b-3p.

ITGA5 is associated with prognosis marker and immunosuppression in head and neck squamous cell carcinoma

BackgroundHead and neck squamous cell carcinoma (HNSCC) is a major tumor that seriously threatens the health of the head and neck or mucosal system. It is manifested as a malignant phenotype of high metastasis and invasion caused by squamous cell transformation in the tissue area. Therefore, it is necessary to search for a biomarker that can systematically correlate and reflect the prognosis of HNSCC based on the characteristics of head and neck tumors.MethodsBased on TCGA-HNSCC data, R software was used to analyze gene expression, correlation, Venn diagram, immune invasive and immunosuppressive phenotypes respectively. The intrinsic effect of ITGA5 on the malignant phenotype of HNSCC cells was verified by cell experiments. Immunohistochemical images from The Human Protein Atlas (THPA) database display the differences in the expression of related proteins in HNSCC tissues. Based on functional enrichment and correlation analysis, the prognostic value of ITGA5 for HNSCC was explored, and the expression level of ITGA5 may affect the chemotherapy of targeting the PI3K-AKT.ResultsIn this study, the target gene ITGA5 may be identified as a valuable prognostic marker for HNSCC. The results of enrichment analysis showed that ITGA5 was mainly involved in the dynamic process of extracellular matrix, which may affect the migration or metastasis of tumor cells. Meanwhile, ITGA5 may be closely related to the infiltration of M2 macrophages, and its secretory phenotypes TGFB1, PDGFA and PDGFB may affect the immunosuppressive phenotypes of tumor cells, which reflects the systemic influence of ITGA5 in HNSCC. In addition, the expression levels of ITGA5 were negatively correlated with the efficacy of targeting PI3K-AKT chemotherapy.ConclusionITGA5 can be used as a potential marker to systematically associate with prognosis of HNSCC, which may be associated with HNSCC malignant phenotype, immunosuppression and chemotherapy resistance.