Vimentin affects colorectal cancer proliferation, invasion, and migration via regulated by activator protein 1.

Uncontrolled recurrence and metastasis are important reasons for the high mortality rate of malignant tumors. Vimentin is positively correlated with the degree of malignancy of cancer cells. Vimentin is also highly expressed in colorectal cancer (CRC) cells and plays a critical role in the metastasis and prognosis of CRC. However, the molecular mechanism of vimentin in the progression of CRC is incompletely understood. Therefore, the most active regions (nucleotides: 785-1085 nt) of the vimentin promoter in CRC were identified using luciferase experiments. By transcription factor sequence search and mutation analysis, the activator protein 1 (AP-1) binding site in the region of 785-1085 nt was confirmed. The vimentin promoter activity was enhanced by overexpression of AP-1. The electrophoretic mobility shift assay and chromatin immunoprecipitation assay showed that the binding site was recognized by AP-1. By cell proliferation assay, colony-forming assay, scratch-wound assay, cell migration assay, and cell invasion assay, we demonstrated that the AP-1 overexpression increased CRC cell proliferation, migration, and invasion. However, when vimentin was knocked down by vimentin small hairpin RNA in the CRC cell of AP-1 overexpression, this trend disappeared. Animal experiments and immunohistochemistry showed that AP-1 promoted tumor growth by regulating the vimentin gene. In summary, AP-1 affected metastasis, invasion of CRC cells in vitro, and tumor growth in vivo by activating the vimentin promoter. This study might provide new insights into the molecular mechanisms of the development of CRC and provide potential therapeutic targets for CRC.

m6 A RNA methylation regulators contribute to malignant progression in rectal cancer.

N6,2'-O-dimethyladenosine (m6 A) RNA methylation, which is correlated with cancer initiation and progression, is dynamically regulated by m6 A RNA methylation regulators, including writers, erasers, and readers. Two subgroups of rectal cancer, including cluster1 and cluster2, were identified based on consensus clustering to m6 A RNA methylation regulators. A protein-protein interaction network was constructed and hub genes were identified. The results demonstrated that the expression of WTAP was significantly associated with YTHDC1 and YTHDF2. The principal component analysis was used to compare the transcriptional profile between cluster1 and cluster2 subgroups. By using two identified m6 A RNA methylation regulators, we constructed a risk signature to predict the survival outcomes of rectal cancer. The results revealed that YTHDC2 and YTHDF2 were protective genes with HR < 1. The coefficients obtained from the least absolute shrinkage and selection operator algorithm were used to calculate the risk score. Patients were then divided into low- and high-risk groups based on the median risk score. The survival analysis demonstrated that there were significant differences in overall survival between these two groups (p < .05). The results of the univariate analysis showed that the risk score, AJCC stage, M stage, and age were associated with overall survival. The results of the multivariate Cox regression analysis showed that the risk score and age were still significantly associated with the overall survival (p < .05). To conclude, m6 A RNA methylation regulators can be regarded as potentially useful biomarkers for predicting the prognosis and designing a treatment strategy in rectal cancer.

Integrative analysis identifies targetable CREB1/FoxA1 transcriptional co-regulation as a predictor of prostate cancer recurrence.

Identifying prostate cancer-driving transcription factors (TFs) in addition to the androgen receptor promises to improve our ability to effectively diagnose and treat this disease. We employed an integrative genomics analysis of master TFs CREB1 and FoxA1 in androgen-dependent prostate cancer (ADPC) and castration-resistant prostate cancer (CRPC) cell lines, primary prostate cancer tissues and circulating tumor cells (CTCs) to investigate their role in defining prostate cancer gene expression profiles. Combining genome-wide binding site and gene expression profiles we define CREB1 as a critical driver of pro-survival, cell cycle and metabolic transcription programs. We show that CREB1 and FoxA1 co-localize and mutually influence each other's binding to define disease-driving transcription profiles associated with advanced prostate cancer. Gene expression analysis in human prostate cancer samples found that CREB1/FoxA1 target gene panels predict prostate cancer recurrence. Finally, we showed that this signaling pathway is sensitive to compounds that inhibit the transcription co-regulatory factor MED1. These findings not only reveal a novel, global transcriptional co-regulatory function of CREB1 and FoxA1, but also suggest CREB1/FoxA1 signaling is a targetable driver of prostate cancer progression and serves as a biomarker of poor clinical outcomes.

Ginsenoside Rg1 improves bone marrow haematopoietic activity via extramedullary haematopoiesis of the spleen.

Cyclophosphamide (CY) is a chemotherapeutic agent used for cancer and immunological diseases. It induces cytotoxicity of bone marrow and causes myelosuppression and extramedullary haematopoiesis (EMH) in treated patients. EMH is characterized with the emergence of multipotent haematopoietic progenitors most likely in the spleen and liver. Previous studies indicated that a Chinese medicine, ginsenoside Rg1, confers a significant effect to elevate the number of lineage (Lin(-) ) Sca-1(+) c-Kit(+) haematopoietic stem and progenitor cells (HSPCs) and restore the function of bone marrow in CY-treated myelosuppressed mice. However, whether the amelioration of bone marrow by Rg1 accompanies an alleviation of EMH in the spleen was still unknown. In our study, the cellularity and weight of the spleen were significantly reduced after Rg1 treatment in CY-treated mice. Moreover, the number of c-Kit(+) HSPCs was significantly decreased but not as a result of apoptosis, indicating that Rg1 alleviated EMH of the spleen induced by CY. Unexpectedly, the proliferation activity of c-Kit(+) HSPCs was only up-regulated in the spleen, but not in the bone marrow, after Rg1 treatment in CY-treated mice. We also found that a fraction of c-Kit(+) /CD45(+) HSPCs was simultaneously increased in the circulation after Rg1 treatment. Interestingly, the effects of Rg1 on the elevation of HSPCs in bone marrow and in the peripheral blood were suppressed in CY-treated splenectomized mice. These results demonstrated that Rg1 improves myelosuppression induced by CY through its action on the proliferation of HSPCs in EMH of the spleen and migration of HSPCs from the spleen to the bone marrow.

DNA methylation screening of primary prostate tumors identifies SRD5A2 and CYP11A1 as candidate markers for assessing risk of biochemical recurrence.

BACKGROUND: Altered DNA methylation in CpG islands of gene promoters has been implicated in prostate cancer (PCa) progression and can be used to predict disease outcome. In this study, we determine whether methylation changes of androgen biosynthesis pathway (ABP)-related genes in patients' plasma cell-free DNA (cfDNA) can serve as prognostic markers for biochemical recurrence (BCR). METHODS: Methyl-binding domain capture sequencing (MBDCap-seq) was used to identify differentially methylated regions (DMRs) in primary tumors of patients who subsequently developed BCR or not, respectively. Methylation pyrosequencing of candidate loci was validated in cfDNA samples of 86 PCa patients taken at and/or post-radical prostatectomy (RP) using univariate and multivariate prediction analyses. RESULTS: Putative DMRs in 13 of 30 ABP-related genes were found between tumors of BCR (n = 12) versus no evidence of disease (NED) (n = 15). In silico analysis of The Cancer Genome Atlas data confirmed increased DNA methylation of two loci-SRD5A2 and CYP11A1, which also correlated with their decreased expression, in tumors with subsequent BCR development. Their aberrant cfDNA methylation was also associated with detectable levels of PSA taken after patients' post-RP. Multivariate analysis of the change in cfDNA methylation at all of CpG sites measured along with patient's treatment history predicted if a patient will develop BCR with 77.5% overall accuracy. CONCLUSIONS: Overall, increased DNA methylation of SRD5A2 and CYP11A1 related to androgen biosynthesis functions may play a role in BCR after patients' RP. The correlation between aberrant cfDNA methylation and detectable PSA in post-RP further suggests their utility as predictive markers for PCa recurrence. .

MicroRNA-21 inhibits p57Kip2 expression in prostate cancer.

BACKGROUND: p57(Kip2), a cyclin-dependent kinase inhibitor, is considered to be a candidate tumor suppressor gene that has been implicated in Beckwith-Wiedemann syndrome and sporadic cancers. In addition, decreased expression of p57(Kip2) protein has been frequently observed in pancreatic, lung, breast, bladder, gastrointestinal tract and prostate cancers. However, p57(Kip2) gene mutations are rare in these cancers suggesting that other unknown mechanisms might be at play in reducing its expression. The aim of this study was to investigate the molecular mechanism of down-regulation of p57(Kip2) in prostate cancer. FINDINGS: We observed a significant negative correlation between the expression of p57(Kip2) and microRNA-21 (miR-21) in prostate cancer samples and after androgen deprivation with castration in the CWR22 human prostate cancer xenograft model. We report that miR-21 targeted the coding region and decreased p57(Kip2) mRNA and protein levels in prostate cancer cells. Conversely, inhibition of endogenous miR-21 by an anti-miR-21 inhibitor strongly induced p57(Kip2) expression. Furthermore, we found that knockdown of p57(Kip2) reversed the effects of the anti-miR-21 inhibitor on cell migration and anchorage-independent cell growth. CONCLUSIONS: Our results indicate that miR-21 is able to downregulate p57(Kip2) expression by targeting the coding region of the gene and is also able to attenuate p57(Kip2) mediated functional responses. This is the first report demonstrating that p57(Kip2) is a novel target of miR-21 in prostate cancer and revealing a novel oncogenic function of this microRNA.

Genome-wide DNA methylation profiling reveals parity-associated hypermethylation of FOXA1.

Early pregnancy in women by the age of 20 is known to have a profound effect on reduction of lifelong breast cancer risk as compared to their nulliparous counterparts. Additional pregnancies further enhance the protection against breast cancer development. Nationwide trend of delayed pregnancy may contribute to the recently reported increase in the incidence of advanced breast cancer among young women in this country. The underlying mechanism for the parity-associated reduction of breast cancer risk is not clearly understood. The purpose of the current study is to use whole-genome DNA methylation profiling to explore a potential association between parity and epigenetic changes in breast tissue from women with early parity and nulliparity. Breast tissue was collected from age-matched cancer-free women with early parity (age < 20; n = 15) or nulliparity (n = 13). The methyl-CpG binding domain-based capture-sequencing technology was used for whole-genome DNA methylation profiling. Potential parity-associated hypermethylated genes were further verified by locus-specific pyrosequencing, using an expanded cohort of parous (n = 19) and nulliparous (n = 16) women that included the initial samples used in the global analysis. Our study identified six genes that are hypermethylated in the parous group (P < 0.05). Pyrosequencing confirmed parity-associated hypermethylation at multiple CpG islands of the FOXA1 gene, which encodes a pioneer factor that facilitates chromatin binding of estrogen receptor α. Our work identifies several potential methylation biomarkers for parity-associated breast cancer risk assessment. In addition, the results are consistent with the notion that parity-associated epigenetic silencing of FOXA1 contributes to long-term attenuation of the estrogenic impact on breast cancer development.

Modulation of YBX1-mediated PANoptosis inhibition by PPM1B and USP10 confers chemoresistance to oxaliplatin in gastric cancer.

Gastric cancer (GC) is a common malignant tumor of the digestive tract, and chemoresistance significantly impacts GC patients' prognosis. PANoptosis has been associated with oxaliplatin-induced cell death. However, the direct regulatory role of YBX1 in cellular chemoresistance through PANoptosis remains unclear. In this study, we investigated the impact of YBX1 on regulating PANoptosis and its influence on the resistance of gastric cancer cells to oxaliplatin. Through overexpression and silencing experiments, we assessed YBX1's effect on proliferation and PANoptosis regulation in gastric cancer cells. Additionally, we identified PPM1B and USP10 as interacting proteins with YBX1 and confirmed their influence on YBX1 molecular function and protein expression levels. Our results demonstrate that YBX1 suppresses PANoptosis, leading to enhanced resistance of gastric cancer cells to oxaliplatin. Furthermore, we found that PPM1B and USP10 play critical roles in regulating YBX1-mediated PANoptosis inhibition. PPM1B directly interacts with YBX1, causing dephosphorylation of YBX1 at serine 314 residue. This dephosphorylation process affects the deubiquitination of YBX1 mediated by USP10, resulting in decreased YBX1 protein expression levels and impacting PANoptosis and oxaliplatin resistance in gastric cancer cells. Additionally, we discovered that the 314th amino acid of YBX1 has a profound impact on its own protein expression abundance, thereby affecting the functionality of YBX1. In conclusion, our study reveals the significance of PPM1B-mediated dephosphorylation of YBX1 and USP10-mediated deubiquitination in regulating PANoptosis and sensitivity to oxaliplatin in gastric cancer cells. These findings offer a potential therapeutic strategy for patients with oxaliplatin-resistant gastric cancer.

Analysis of the characteristics of immune infiltration in endometrial carcinoma and its relationship with prognosis based on bioinformatics.

To explore immune-related molecules that affect the prognosis of endometrial carcinoma (EC) using bioinformatic data mining. The expression data related to EC were downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus databases. After differential expression analysis, the intersection with immune related genes in the ImmPort database was used to obtain immune related differentially expressed genes (IRDEGs). The correlation between clinicopathological information and the prognosis of IRDEGs was further analyzed to obtain prognosis related differentially expressed immune genes (PRDEIG). Gene correlation analysis and Gene Set Enrichment Analysis (GSEA) enrichment analysis showed that PRDEIG was enriched in cancer-related functional pathways. We then analyzed the relationship between PRDEIG and immune cell infiltration, and further analyzed the mRNA and protein expression of PRDEIG in EC using TCGA and the human protein expression atlas (THPA) databases. After the intersection of the differential expression analysis results and immune-related genes, 4 IRDEGs were obtained: osteoglycin (OGN), LTBP4, CXCL12, and SPP1. After analyzing the relationship between 4 IRDEGs and clinicopathological parameters and prognosis of patients with EC, revealed that only OGN was not only related to tumor immunity, but also affected the prognosis of patients with EC. Gene correlation and GSEA enrichment of OGN were analyzed. The results showed that OGN was significantly enriched in 6 functional pathways: epithelial mesenchymal transition, KRAS signaling up, myogenesis, UV response, allograft rejection and apical junction. In addition, it was also found that OGN was significantly correlated with a variety of immune cells. The results of TCGA and THPA database showed that the mRNA and protein expression levels of OGN decreased in EC. OGN may affect the epithelial mesenchymal transformation (EMT) of tumor by affecting the infiltration of tumor immune cells.

Screening of hub inflammatory bowel disease biomarkers and identification of immune-related functions based on basement membrane genes.

BACKGROUND: Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), is a chronic, inflammatory, and autoimmune disease, but its specific etiology and pathogenesis are still unclear. This study aimed to better discover the causative basement membrane (BM) genes of their subtypes and their associations. METHODS: The differential expression of BM genes between CD and UC was analyzed and validated by downloading relevant datasets from the GEO database. We divided the samples into 3 groups for comparative analysis. Construction of PPI networks, enrichment of differential gene functions, screening of Lasso regression models, validation of ROC curves, nomogram for disease prediction and other analytical methods were used. The immune cell infiltration was further explored by ssGSEA analysis, the immune correlates of hub BM genes were found, and finally, the hub central genes were screened by machine learning. RESULTS: We obtained 6 candidate hub BM genes related to cellular immune infiltration in the CD and UC groups, respectively, and further screened the central hub genes ADAMTS17 and ADAMTS9 through machine learning. And in the ROC curve models, AUC > 0.7, indicating that this characteristic gene has a more accurate predictive effect on IBD. We also found that the pathogenicity-related BM genes of the CD and UC groups were mainly concentrated in the ADAMTS family (ADAMTS17 and ADAMTS9). Addition there are some differences between the two subtypes, and the central different hub BM genes are SPARC, POSTN, and ADAMTS2. CONCLUSIONS: In the current study, we provided a nomogram model of CD and UC composed of BM genes, identified central hub genes, and clarified the similarities and differences between CD and UC. This will have potential value for preclinical, clinical, and translational guidance and differential research in IBD.

SLC26A3/NHERF2-IκB/NFκB/p65 feedback loop suppresses tumorigenesis and metastasis in colorectal cancer.

Colorectal cancer (CRC) is a formidable disease due to the intricate mechanisms that drive its proliferation and metastasis. Despite significant progress in cancer research, the integration of these mechanisms that influence cancer cell behavior remains elusive. Therefore, it is imperative to comprehensively elucidate the underlying mechanisms driving CRC proliferation and metastasis. In this study, we reported a novel role of SLC26A3 in suppressing CRC progression. We found that SLC26A3 expression was downregulated in CRC, which was proportionally correlated with survival. Our in vivo and in vitro experiments demonstrated that up-regulation of SLC26A3 inhibited CRC proliferation and metastasis, while down-regulation of SLC26A3 promoted CRC progression by modulating the expression level of IκB. Furthermore, we identified NHERF2 as a novel interacting protein of SLC26A3 responsible for stabilizing the IκB protein and removing ubiquitination modification. Mechanistically, SLC26A3 augmented the interaction between NHERF2 and IκB, subsequently reducing its degradation. This process inhibited the dissociation of p65 from the IκB/p65/p50 complex and reduced the translocation of p65 from the cytoplasm to the nucleus. Moreover, our investigation revealed that NF-κB/p65 directly bound to the promoter of SLC26A3, leading to a decline in its mRNA expression. Thus, SLC26A3 impeded the nuclear translocation of NF-κB/p65, enhancing the transcription of SLC26A3 and establishing a positive regulatory feedback loop in CRC cells. Collectively, these results suggest that a SLC26A3/NHERF2-IκB/NF-κB/p65 signaling loop suppresses proliferation and metastasis in CRC cells. These findings propose a novel SLC26A3-driven signaling loop that regulates proliferation and metastasis in CRC, providing promising therapeutic interventions and prognostic targets for the management of CRC.

Bioinformatics Analysis of Immune Infiltration In Endometrial Carcinoma Tumors and its Relationship With Prognosis.

BACKGROUND/AIM: Endometrial carcinoma (EC) is the most common gynecological cancer, but lacks specific targetable markers. In order to explore the immune-related molecules that affect the progression and prognosis of EC, we analyzed the differential expression of genes in different histological grades of the disease. MATERIALS AND METHODS: EC-related gene-expression data of different histological grades were downloaded from TCGA and GEO databases. The list of immune-related genes was obtained from the ImmPort database. In order to identity differentially-expressed genes (DEGs), differential-expression analysis was performed. The intersection of DEGs and immune-related genes was termed immune-related differentially-expressed genes (IRDEGs). IRDEGs were enriched in cancer-related functional pathways by gene-correlation analysis and GSEA-enrichment analysis. The association of IRDEGs with immune-cell tumor infiltration and gene polymorphisms was analyzed using IRDEG mRNA and protein-expression data in EC from TCGA and THPA databases. RESULTS: Three IRDEGs, TNFSF15, SEMA3E and TNFSF10, were involved in the analysis of the prognosis of EC patients. IRDEGs were not only related to clinical characteristics but could also affect the prognosis of patients. Gene-correlation and GSEA-enrichment analysis of IRDEGs showed that TNFSF15 and TNFSF10 were co-enriched in the IL2-STAT5 functional pathway. IRDEGs had a significant correlation with a variety of immune-cell types infiltrating EC tumors and were related to EC prognosis. IRDEG mRNA- and protein-expression levels were increased in EC compared to normal tissues. CONCLUSION: TNFSF15, SEMA3E and TNFSF10 may regulate the progression and prognosis of EC patients by affecting immune-cell infiltration of EC tumors.

TRAF6 regulates the abundance of RIPK1 and inhibits the RIPK1/RIPK3/MLKL necroptosis signaling pathway and affects the progression of colorectal cancer.

Colorectal cancer cannot be completely cured at present, and it is still an important clinical medical problem. TRAF6 is highly expressed in many malignant tumors. However, the role of TRAF6 in colorectal cancer is still controversial, mainly because the specific regulatory mechanism of colorectal cancer is still unclear, and the death mode of colorectal cancer cells has not been elucidated. The recent study found that TRAF6 inhibits necroptosis in colorectal cancer cells via the RIPK1/RIPK3/MLKL signaling pathway. The RIPK1 inhibitor Necrostain-1 inhibits colorectal cancer cell necroptosis via the RIPK1/RIPK3/MLKL signaling pathway. TRAF6 directly interacts with RIPK1 through the polyubiquitination of Lys48-linked RIPK1 and reduces the levels of RIPK1 protein in colorectal cancer cells, leading to necroptosis, thus promoting the proliferation of colorectal cancer cells. The recent study demonstrated that TRAF6 promotes colorectal cell progression by inhibiting the RIPK1/RIPK3/MLKL necroptosis signaling pathway, which may provide a new therapeutic target for colorectal cancer.

KLRF1, a novel marker of CD56bright NK cells, predicts improved survival for patients with locally advanced bladder cancer.

BACKGROUND: Bladder tumor-infiltrating CD56bright NK cells are more tumor cytotoxic than their CD56dim counterparts. Identification of NK cell subsets is labor-intensive and has limited utility in the clinical setting. Here, we sought to identify a surrogate marker of bladder CD56bright NK cells and to test its prognostic significance. METHODS: CD56bright and CD56dim NK cells were characterized with the multiparametric flow (n = 20) and mass cytometry (n = 21) in human bladder tumors. Transcriptome data from bladder tumors (n = 351) profiled by The Cancer Genome Atlas (TCGA) were analyzed. The expression levels of individual markers in intratumoral CD56bright and CD56dim NK cells were visualized in tSNE plots. Expressions of activation markers were also compared between Killer Cell Lectin-Like Receptor Subfamily F Member 1 (KLRF1)+ and KLRF1- NK cells. RESULTS: Intratumoral CD56bright NK cells displayed a more activated phenotype compared to the CD56dim subset. Multiple intratumoral cell types expressed CD56, including bladder tumor cells and nonspecific intratumoral CD56 expression was associated with worse patient survival. Thus, an alternative to CD56 as a marker of CD56bright NK cells was sought. The activation receptor KLRF1 was significantly increased on CD56bright but not on CD56dim NK cells. Intratumoral KLRF1+ NK cells were more activated and expressed higher levels of activation molecules compared with KLRF1- NK cells, analogous to the distinct effector function of NK cells across CD56 expression. High intratumoral KLRF1 was associated with improved recurrence-free survival (hazard ratio [HR] 0.53, p = 0.01), cancer-specific survival (HR 0.47, p = 0.02), and overall survival (HR 0.54, p = 0.02) on multivariable analyses that adjusted for clinical and pathologic variables. CONCLUSIONS: KLRF1 is a promising prognostic marker in bladder cancer and may guide treatment decisions upon validation.

2-Hydroxyglutarate destabilizes chromatin regulatory landscape and lineage fidelity to promote cellular heterogeneity.

The epigenome delineates lineage-specific transcriptional programs and restricts cell plasticity to prevent non-physiological cell fate transitions. Although cell diversification fosters tumor evolution and therapy resistance, upstream mechanisms that regulate the stability and plasticity of the cancer epigenome remain elusive. Here we show that 2-hydroxyglutarate (2HG) not only suppresses DNA repair but also mediates the high-plasticity chromatin landscape. A combination of single-cell epigenomics and multi-omics approaches demonstrates that 2HG disarranges otherwise well-preserved stable nucleosome positioning and promotes cell-to-cell variability. 2HG induces loss of motif accessibility to the luminal-defining transcriptional factors FOXA1, FOXP1, and GATA3 and a shift from luminal to basal-like gene expression. Breast tumors with high 2HG exhibit enhanced heterogeneity with undifferentiated epigenomic signatures linked to adverse prognosis. Further, ascorbate-2-phosphate (A2P) eradicates heterogeneity and impairs growth of high 2HG-producing breast cancer cells. These findings suggest 2HG as a key determinant of cancer plasticity and provide a rational strategy to counteract tumor cell evolution.

Bladder tumor ILC1s undergo Th17-like differentiation in human bladder cancer.

PURPOSE: Human innate lymphoid cells (hILCs) are lineage-negative immune cells that do not express rearranged adaptive antigen receptors. Natural killer (NK) cells are hILCs that contribute to cancer defense. The role of non-NK hILCs in cancer is unclear. Our study aimed to characterize non-NK hILCs in bladder cancer. EXPERIMENTAL DESIGN: Mass cytometry was used to characterize intratumoral non-NK hILCs based on 35 parameters, including receptors, cytokines, and transcription factors from 21 muscle-invasive bladder tumors. Model-based clustering was performed on t-distributed stochastic neighbor embedding (t-SNE) coordinates of hILCs, and the association of hILCs with tumor stage was analyzed. RESULTS: Most frequent among intratumoral non-NK hILCs were hILC1s, which were increased in higher compared with lower stage tumors. Intratumoral hILC1s were marked by Th17-like phenotype with high RORγt, IL-17, and IL-22 compared to Th1 differentiation markers, including Tbet, perforin, and IFN-γ. Compared with intratumoral hILC2s and hILC3s, hILC1s also had lower expression of activation markers (NKp30, NKp46, and CD69) and increased expression of exhaustion molecules (PD-1 and Tim3). Unsupervised clustering identified nine clusters of bladder hILCs, which were not defined by the primary hILC subtypes 1-3. hILC1s featured in all the nine clusters indicating that intratumoral hILC1s displayed the highest phenotypic heterogeneity among all hILCs. CONCLUSIONS: hILC1s are increased in higher stage tumors among patients with muscle-invasive bladder cancer. These intratumoral hILC1s exhibit an exhausted phenotype and Th17-like differentiation, identifying them as potential targets for immunotherapy.

Contacts with Macrophages Promote an Aggressive Nanomechanical Phenotype of Circulating Tumor Cells in Prostate Cancer.

Aggressive tumors of epithelial origin shed cells that intravasate and become circulating tumor cells (CTC). The CTCs that are able to survive the stresses encountered in the bloodstream can then seed metastases. We demonstrated previously that CTCs isolated from the blood of prostate cancer patients display specific nanomechanical phenotypes characteristic of cell endurance and invasiveness and patient sensitivity to androgen deprivation therapy. Here we report that patient-isolated CTCs are nanomechanically distinct from cells randomly shed from the tumor, with high adhesion as the most distinguishing biophysical marker. CTCs uniquely coisolated with macrophage-like cells bearing the markers of tumor-associated macrophages (TAM). The presence of these immune cells was indicative of a survival-promoting phenotype of "mechanical fitness" in CTCs based on high softness and high adhesion as determined by atomic force microscopy. Correlations between enumeration of macrophages and mechanical fitness of CTCs were strong in patients before the start of hormonal therapy. Single-cell proteomic analysis and nanomechanical phenotyping of tumor cell-macrophage cocultures revealed that macrophages promoted epithelial-mesenchymal plasticity in prostate cancer cells, manifesting in their mechanical fitness. The resulting softness and adhesiveness of the mechanically fit CTCs confer resistance to shear stress and enable protective cell clustering. These findings suggest that selected tumor cells are coached by TAMs and accompanied by them to acquire intermediate epithelial/mesenchymal status, thereby facilitating survival during the critical early stage leading to metastasis. SIGNIFICANCE: The interaction between macrophages and circulating tumor cells increases the capacity of tumor cells to initiate metastasis and may constitute a new set of blood-based targets for pharmacologic intervention.

MyD88 mediates colorectal cancer cell proliferation, migration and invasion via NF­κB/AP­1 signaling pathway.

The role of myeloid differentiation factor 88 (MyD88) in malignant tumors is largely unknown. Therefore, in this study, we aimed to examine the function and underlying mechanism of MyD88 in colorectal carcinoma in vitro using SW480 and HCT116 cell lines and in vivo using a nude mouse model. SW480 and HCT116 cells were infected with a lentiviral­based effective MyD88 siRNA virus. CCK­8 and colony formation assay were used to assess cell proliferation. Transwell and scratch assays were used to test the migration of colorectal cancer cells, and the Transwell assay was further used to analyze the invasiveness of colorectal cancer cells. Western blotting was performed to analyze the underlying mechanism of MyD88 regulation. In vitro experiments demonstrated that silencing MyD88 in SW480 and HCT116 cells markedly suppressed growth and invasion. Furthermore, MyD88 knockdown affected the MyD88­NF­κB/AP­1 signaling pathways in SW480 and HCT116 cells. In vivo, MyD88 knockdown inhibited tumor growth in a HCT116 cell subcutaneous nude model. We found that knockdown of the MyD88 gene can affect proliferation, invasion, and migration of colorectal cancer cells. We further verified that MyD88 knockdown can reduce the activity of NF­κB and AP­1 pathways. These results show that MyD88 gene plays an important role in promoting colorectal cancer, and thus can be exploited as a potential diagnostic and prognostic biomarker for colorectal cancer.

The Effects of TRAF6 on Growth and Progression in Colorectal Cancer are Regulated by miRNA-140.

BACKGROUND AND AIM: Some studies have confirmed that miRNA-140 exhibits a suppressive role in gastric cancer, Wilms' tumor. However, the function of miRNA-140 in colorectal cancer has not been completely elucidated. The present study aims to verify TRAF6 as the targeted gene by miRNA-140 which was investigated in colorectal cancer tissues and cells, and its effects on the biological characteristics of colorectal cancer cells were determined, in order to provide an experimental and theoretical basis for the application of TRAF6 in the treatment of colorectal cancer. METHODS: qPCR analyzed miRNA-140 expression levels in colorectal cancer tissues, normal colorectal cancer tissues and colorectal cells including SW480 and HCT116 cancer cells and FHC normal colorectal epithetical cells. A serial biological experiment analyzed miRNA-140 effects on cell proliferation, migration and invasion capacities in SW480 and HCT116 cells. miRNA targeting gene prediction and a dual luciferase assay were used to analyze miRNA-140-targeted TRAF6. qPCR and Western blot analyzed miRNA-140 effects on the mRNA and protein expression of TRAF6. Western blot analyzed miRNA-140 effects on NF-κB/c-jun signaling pathways. Animal studies were performed to investigate the effects of miRNA-140 on colorectal cancer implantation tumor growth. Immunohistochemistry analyzed TRAF6 expression in animal experimentation tumors. RESULTS: miRNA-140 expression is lower in colorectal cancer tissues and colorectal cancer cells. Over-expression of miRNA-140 inhibited the proliferation, migration and invasion capacities of colorectal cancer cells. miRNA-140 targeted the TRAF6 mRNA 3'UTR area and decreased TRAF6 protein expression. miRNA-140 suppressed p-NF-κB/p-c-jun proteins expression. miRNA-140 inhibited colorectal cancer implantation tumor growth in the mice model. CONCLUSION: miRNA-140 targeting TRAF6 affects the progression and growth of colorectal cancer, the mechanism could be miRNA-140 decreasing the TRAF6 expression effects on the NF-κB/c-jun signaling pathways.