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Breast cancer is one of the most serious and deadly cancers in women worldwide, with distant metastases being the leading cause of death. Tn antigen, a tumor‐associated carbohydrate antigen, was frequently detected in breast cancer, but its exact role in breast cancer metastasis has not been well elucidated. Here we investigated the impact of Tn antigen expression on breast cancer metastasis and its underlying mechanisms. The expression of Tn antigen was induced in two breast cancer cell lines by deleting T‐synthase or Cosmc, both of which are required for normal O‐glycosylation. It showed that Tn‐expressing cancer cells promoted epithelial–mesenchymal transition (EMT) and metastatic features as compared to Tn(−) control cells both in vitro and in vivo. Mechanistically, we found that cancer susceptibility candidate 4 (CASC4), a heavily O‐glycosylated protein, was significantly downregulated in both Tn(+) cells. Overexpression of CASC4 suppressed Tn‐induced activation of EMT and cancer metastasis via inhibition of Cdc42 signaling. Furthermore, we confirmed that O‐glycosylation is essential for the functional role of CASC4 because defective O‐glycosylated CASC4 (mutant CASC4, which lacks nine O‐glycosylation sites) exerted marginal metastatic‐suppressing effects in comparison with WT CASC4. Collectively, these data suggest that Tn‐mediated aberrant O‐glycosylation contributes to breast cancer metastasis via impairment of CASC4 expression and function. [ABSTRACT FROM AUTHOR]

Ovarian cancer (OC) is a particularly lethal disease due to intratumoral heterogeneity, resistance to traditional chemotherapy, and poor response to targeted therapy and immunotherapy. Interferon-γ (IFN-γ) is an attractive therapeutic cytokine, with positive responses achieved in multiple OC clinical trials. However, clinical application of IFN-γ in OC is still hindered, due to the severe toxicity when used at higher levels, as well as the considerable pro-metastatic adverse effect when used at lower levels. Thus, an effective combined intervention is needed to enhance the anti-tumor efficacy of IFN-γ and to suppress the IFN-γ-induced metastasis. Here, we uncovered that OC cells develop an adaptive strategy by upregulating midkine (MDK) to counteract the IFN-γ-induced anti-tumor activity and to fuel IFN-γ-induced metastasis. We showed that MDK is a critical downstream target of IFN-γ in OC, and that this regulation acts in a dose-dependent manner and is mediated by STAT1. Gain-of-function studies showed that MDK overexpression promotes cell proliferation and metastasis in OC, indicating that IFN-γ-activated MDK may antagonize IFN-γ in inhibiting OC proliferation but synergize IFN-γ in promoting OC metastasis. Subsequently, we assessed the influence of MDK inhibition on IFN-γ-induced anti-proliferation and pro-metastasis effects using an MDK inhibitor (iMDK), and we found that MDK inhibition robustly enhanced IFN-γ-induced growth inhibition (all CIs < 0.1) and reversed IFN-γ-driven epithelial-to-mesenchymal transition (EMT) and metastasis in OC in vitro. Collectively, these data identify an IFN-γ responsive protein, MDK, in counteracting anti-proliferation while endowing the pro-metastatic role of IFN-γ in cancer treatment, and we therefore propose the combined utilization of the MDK inhibitor in IFN-γ-based therapies in future OC treatment. [ABSTRACT FROM AUTHOR]

IFN-γ is a pleiotropic cytokine with immunomodulatory and tumoricidal functions. It has been used as an anti-tumor agent in adjuvant therapies for various cancers. Paradoxically, recent advances have also demonstrated pro-tumorigenic effects of IFN-γ, especially in promoting cancer metastasis, with the mechanism remains unclear. This will undoubtedly hinder the application of IFN-γ in cancer treatment. Here, we verified that IFN-γ treatment led to activation of the epithelial-to-mesenchymal transition (EMT) programme and metastasis in cell lines of various cancers, including the kidney cancer cell line Caki-1, the lung cancer cell line A549, the cervical carcinoma cell line CaSki, the breast cancer cell line BT549 and the colon cancer cell line HCT116. We further disclosed that midkine (MDK), an emerging oncoprotein and EMT inducer, is a common responsive target of IFN-γ in these cell lines. Mechanistically, IFN-γ upregulated MDK via STAT1, a principle downstream effector in the IFN-γ signalling. MDK is elevated in the majority of cancer types in the TCGA database, and its overexpression drove EMT activation and cancer metastasis in all examined cell lines. Targeting MDK using a specific MDK inhibitor (iMDK) broadly reversed IFN-γ-activated EMT, and subsequently abrogated IFN-γ-triggered metastasis. Collectively, our data uncover a MDK-dependent EMT inducing mechanism underlying IFN-γ-driven metastasis across cancers which could be attenuated by pharmacological inhibition of MDK. Based on these findings, we propose that MDK may be used as a potential therapeutic target to eliminate IFN-γ-elicited pro-metastatic adverse effect, and that combined MDK utilization may expand the application of IFN-γ in cancer and improve the clinical benefits from IFN-γ-based therapies. [ABSTRACT FROM AUTHOR]

Nicotinamide nucleotide transhydrogenase-antisense RNA1 (NNT-AS1) is a long noncoding RNA (lncRNA) that has been shown to be overexpressed in hepatocellular carcinoma (HCC). However, the molecular mechanism involving NNT-AS1 in HCC remains to be extensively investigated. The activation of TGF-β signaling inhibits tumor-infiltrating lymphocytes (TILs) and results in tumor immune evasion. We thus planned to explore the mechanism by which NNT-AS1 activates the TGF-β signaling pathway and inhibits TILs in HCC. High levels of NNT-AS1 were detected in HCC tissues by both RNAscope and real-time quantitative PCR (RT-qPCR) assays. The levels of proteins involved in TGF-β signaling and those of CD4 T lymphocytes were quantified by immunohistochemistry (IHC). HCC cell lines (HepG2 and Huh7) were used to explore the effects of NNT-AS1 on TGF-β signaling activation. In these analyses, RNAscope detection demonstrated that NNT-AS1 levels were significantly increased in HCC cancer tissues (P = 0.0001). In addition, the elevated NNT-AS1 levels in cancer tissue were further confirmed by RT-qPCR analysis of HCC cancer tissues (n = 64) and normal tissues (n = 26) (P = 0.0003). Importantly, the overall survival time of HCC patients who exhibited higher levels of NNT-AS1 expression was significantly shorter than that of HCC patients who had lower levels of NNT-AS1 expression (P = 0.0402). Further mechanistic investigation indicated that NNT-AS1 inhibition significantly decreased the levels of TGF-β, TGFBR1, and SMAD5 in HCC cells. In HCC tissues, IHC detection showed that relatively high NNT-AS1 levels were associated with a reduction in infiltrated CD4 lymphocyte numbers. In conclusion, this research identifies a novel mechanism by which NNT-AS1 impairs CD4 T cell infiltration via activation of the TGF-β signaling pathway in HCC. [ABSTRACT FROM AUTHOR]

Cosmc is known as a T‐synthase‐specific molecular chaperone that plays a crucial role in the process of O‐glycosylation. Cosmc dysfunction leads to inactive T‐synthase and results in aberrant O‐glycosylation, which is associated with various tumour malignancies. However, it is unclear whether Cosmc has some other functions beyond its involvement in O‐glycosylation. In this study, we aimed to investigate the functional role of Cosmc in human colorectal cancer (CRC). We first assessed the expression levels of Cosmc in human CRC specimens and then forcedly expressed Cosmc in human CRC cell lines (HCT116, SW480) to examine its impact on cellular behaviours. The mechanisms for aberrant expression of Cosmc in CRC tissues and the altered behaviours of tumour cells were explored. It showed that the mRNA and protein levels of Cosmc were markedly elevated in human CRC specimens relative to normal colorectal tissues. The occurrence of endoplasmic reticulum (ER) stress may largely contribute to the increased Cosmc expression in cancer tissue and cells. Cosmc overexpression in CRC cells significantly promoted cell migration and invasion, which could be attributed to the activation of the epithelial‐mesenchymal transition (EMT) pathway rather than aberrant O‐glycosylation. These data indicate that Cosmc expression was elevated in human CRC possibly caused by ER stress, which further enhanced malignancies through the activation of EMT but independently of aberrant O‐glycosylation. [ABSTRACT FROM AUTHOR]

Background: Breast cancer remains the most lethal malignancy in women worldwide. Aberrant O-glycosylation is closely related to many human diseases, including breast carcinoma; however, its precise role in cancer development is insufficiently understood. Cosmc is an endoplasmic reticulum-localized chaperone that regulates the O-glycosylation of proteins. Cosmc dysfunction results in inactive T-synthase and expression of truncated O-glycans such as Tn antigen. Here we investigated the impact of Cosmc disruption-mediated aberrant O-glycosylation on breast cancer cell development through in vitro and in vivo experiments. Materials and Methods: We deleted the Cosmc gene in two breast cancer cell lines (MCF7, T47D) using the CRISPR/Cas-9 system and then measured the expression levels of Tn antigen. The proliferation of Tn-positive cells was examined by RTCA, colony formation and in vivo experiments. The effects of Cosmc deficiency on glycoprotein CD44 and MAPK pathway were also determined. Results: Both in vitro and in vivo studies showed that Cosmc deficiency markedly suppressed breast cancer cell growth compared with the corresponding controls. Mechanistically, Cosmc disruption impaired the protein expression of CD44 and the associated MAPK signaling pathway; the latter plays a crucial role in cell proliferation. Reconstitution of CD44 substantially reversed the observed alterations, confirming that CD44 requires normal O-glycosylation for its proper expression and activation of the related signaling pathway. Conclusion: This study showed that Cosmc deficiency-mediated aberrant O-glycosylation suppressed breast cancer cell growth, which was likely mediated by the impairment of CD44 expression. [ABSTRACT FROM AUTHOR]

Tn antigen is a truncated O‐glycan, frequently detected in colorectal cancer (CRC), but its precise role in CRC metastasis is not well addressed. Here we investigated the effects of Core 1 β3Gal‐T specific molecular chaperone (Cosmc) deletion‐mediated Tn antigen exposure on CRC metastasis and its underlying mechanism. We first used CRISPR/Cas9 technology to knockout Cosmc, which is required for normal O‐glycosylation, and thereby obtained Tn‐positive CRC cells. We then investigated the biological consequences of Tn antigen expression in CRC. The results showed that Tn‐positive cells exhibited an enhanced metastatic capability both in vitro and in vivo. A further analysis indicated that Tn antigen expression induced typical activation of epithelial‐mesenchymal transition (EMT). Mechanistically, we found that H‐Ras, which is known to drive EMT, was markedly up‐regulated in Tn‐positive cells, whereas knockdown of H‐Ras suppressed Tn antigen induced activation of EMT. Furthermore, we confirmed that LS174T cells (Tn‐positive) transfected with wild‐type Cosmc, thus expressing no Tn antigen, had down‐regulation of H‐Ras expression and subsequent inhibition of EMT process. In addition, analysis of 438 samples in TCGA cohort demonstrated that Cosmc expression was reversely correlated with H‐Ras, underscoring the significance of Tn antigen‐H‐Ras signalling in CRC patients. These data demonstrated that Cosmc deletion‐mediated Tn antigen exposure promotes CRC metastasis, which is possibly mediated by H‐Ras‐induced EMT activation. [ABSTRACT FROM AUTHOR]

Abstract: Aberrant O‐glycosylation is frequently observed in colorectal cancer (CRC) patients, but it is unclear if it contributes intrinsically to tumorigenesis. Here, we investigated the biological consequences of aberrant O‐glycosylation in CRC. We first detected the expression profile of Tn antigen in a serial of human CRC tissues and then explored the genetic and biosynthetic mechanisms. Moreover, we used a human CRC cell line (LS174T), which express Tn antigen, to assess whether aberrant O‐glycosylation can directly promote oncogenic properties. It showed that Tn antigen was detected in around 86% human primary and metastatic CRC tissues. Bio‐functional investigations showed that T‐synthase and Cosmc were both impaired in cancer tissues. A further analysis detected an occurrence of hypermethylation of Cosmc gene, which possibly caused its loss‐of‐function and a consequent inactive T‐synthase. Transfection of LS174T cells with WT Cosmc restored mature O‐glycosylation, which subsequently down‐regulated cancer cell proliferation, migration and apoptotic‐resistant ability. Significantly, the expression of MUC2, a heavily O‐glycosylated glycoprotein that plays an essential role in intestinal function, was uniformly reduced in human CRC tissues as well as in LS174T cells. These data suggest that aberrant O‐glycosylation contributes to the development of CRC through direct induction of oncogenic properties in cancer cells. [ABSTRACT FROM AUTHOR]

Background. Immature truncated O-glycans such as Tn antigen are frequently detected in human colorectal cancer (CRC); however, the precise pathological consequences of Tn antigen expression on CRC are unknown. T-synthase is the key enzyme required for biosynthesis of mature O-glycans. Here we investigated the functional roles of Tn antigen expression mediated by T-synthase deficiency in CRC cells. Methods. To knock out T-synthase, we used CRISPR-Cas9 technology to target C1GALT1, the gene encoding T-synthase, in a CRC cell line (HCT116). Deletion of T-synthase was confirmed by western blotting, and expression of Tn antigen was determined by flow cytometry in HCT116 cells. We then assessed the biological effects of T-synthase deficiency on oncogenic behaviors in HCT116 cells. Furthermore, we analyzed the mechanistic role of T-synthase deficiency in cancer cells by determining the epithelial-mesenchymal transition (EMT) pathway. Results. We showed that forced knockout of T-synthase in HCT116 cells significantly induced Tn antigen expression, which represented the occurrence of aberrant O-glycosylation. Loss of T-synthase significantly enhanced cell proliferation and adhesion, as well as migration and invasiveness in culture. More importantly, we demonstrated that T-synthase deficiency directly induced classical EMT characteristics in cancer cells. E-cadherin, a typical epithelial cell marker, was markedly decreased in T-synthase knockout HCT 116 cells, accompanied by an enhanced expression of mesenchymal markers including snail and fibronectin (FN). Conclusions. These findings indicate that T-synthase deficiency in CRC cells not only is responsible for aberrant O-glycosylation, but also triggers the molecular process of EMT pathway, which may translate to increased invasiveness and metastasis in cancers. [ABSTRACT FROM AUTHOR]

Aberrant O-glycosylation truncates O-glycans and is known to be closely associated with colorectal cancer (CRC), a major gastrointestinal tumor. CD44 is one of the highly post-transcriptionally modified O-glycoproteins participating in a series of physiological and pathobiological processes. In this research, we aimed to investigate whether CD44 expression in cells and exosomes can be influenced by disruption of Core 1-mediated O-glycosylation. Exosomes derived from LS174T and LSC human colon cancer cell lines were isolated from cell culture supernatant and pulled down using tetraspanin-specific antibody CD63 immunoaffinity magnetic beads. Identifications have been performed via transmission electron microscopy (TEM) and flow cytometry. CD63 immunoaffinity-purified exosomes are examined for CD44 expression by flow cytometric analyses. The percentages of CD44 in exosomes derived from abnormally O-glycosylated cells are significantly higher compared with those derived from normal ones, however, which is surprisingly contrary to the cellular expression levels of CD44. The secretion of truncated glycoproteins to the extracellular environment via microvesicles may be most likely its underlying mechanism. CD44 in exosomes might be a potential biomarker of aberrant O-glycosylation. This is the first study indicating that aberrant O-glycosylation can affect expression or delivery of O-glycoproteins via exosomes, which provides us some new sights in therapeutic strategies for human colon cancer. • Aberrant Core 1-mediated O-glycosylation down-regulates intracellular CD44 protein expression while up-regulates its exosomal expression. • Complementation with wtCosmc restores abnormal O-glycosylation, thereby corrects its effects on CD44 both in cells and exosomes. • Truncated CD44 is released to the extracellular environment via exosomes. [ABSTRACT FROM AUTHOR]

• IFNAR1 expression in CD4+ and CD8+ T cells is higher in CRC. • Peripheral blood CD4+ T IFNAR1 levels positively associate with TME CD8+ T cell infiltration. • CD8+ T IFNAR1 expressions were negative associated with plasma IL-2, IFN-γ, and TNFα. • IFNAR1 levels in peripheral blood CD8+ T negatively associate with chemosensitivity. IFN-α receptor (IFNAR) is critical for maintaining the crosstalk between cancer cells and lymphocytes. We investigated IFNAR1 expression in peripheral blood CD4+ and CD8+ T cells and explored their relationships with plasma cytokines, chemosensitivity and infiltrated T cells in the tumor microenvironment (TME) of colorectal cancer (CRC). The levels of IFNAR1, IFN-γ, and PD1 in peripheral T cells were tested using flow cytometry. Immunohistochemical staining of IFNAR1 in CRC tissues was performed. A cytometric bead array was used to determine the plasma concentrations of cytokines. In CRC patients, IFNAR1 levels were significantly increased in peripheral blood T cells, and plasma IL-6 levels were also significantly increased. Pearson correlation analysis revealed that IFNAR1 expression in CD8+ T cells was negatively associated with plasma IL-2, IFN-γ, and TNFα. IFNAR1 expression in CD4+ T cells was positively associated with TME infiltrated levels of CD8+ T cells. The levels of CD8+ T cells with IFNAR1 and plasma IFN-γ were associated with chemosensitivity. Collectively, IFNAR1 levels in CD4+ and CD8+ T cells were significantly upregulated in CRC patients and positively associated with T-cell infiltration. IFNAR1 may be a chemotherapy biomarker for predicting response. [ABSTRACT FROM AUTHOR]

Background: The objective of this study is to study the adverse effects of coal mining environment on workers to discover early effective biomarkers., Methods: The molecular epidemiological study was conducted with 502 in-service workers, who were divided into miner and auxiliary. We measured the individual levels of dust exposure for participants. Clinical examinations were conducted by qualified doctors. Peripheral blood was collected to measure biochemistry, hemogram, and karyocyte apoptosis., Results: All workers were healthy who have not found with any diseases that can be diagnosed medically in the physical examination and showed no difference in dust exposure level, age, height, weight, and body mass index between groups. The working years of miners were lower than that of auxiliaries ( p  < 0.001). Compared with auxiliaries, the concentration and percentage of lymphocytes ( p  = 0.040, p  = 0.012), basophils ( p  = 0.027, p  = 0.034), and red blood cells ( p  < 0.001) and the concentration of hemoglobin of miners were lower ( p  < 0.001). The percentage of neutrophils ( p  = 0.003), the concentration of mean corpuscular hemoglobin concentration ( p  = 0.002), and the proportion of karyocyte apoptosis in miners were higher ( p < 0.001). Miners presented higher blood urea nitrogen ( p  < 0.001), ratio of blood urea nitrogen to creatinine ( p  < 0.001), the high density lipoprotein cholesterol ( p  < 0.001), lower creatinine ( p  < 0.05), and cholesterol ( p  < 0.001)., Conclusion: The coal mining environment impacted mining workers' immune function, renal function, and the hematopoietic system, including BUN/CRE, HGB, RBC, and LYMPH, which could be used as early biomarkers to screen the health of coal miners., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Chen, Ding, Zhang and Dong.)

Ovarian cancer (OC) is a particularly lethal disease due to intratumoral heterogeneity, resistance to traditional chemotherapy, and poor response to targeted therapy and immunotherapy. Interferon-γ (IFN-γ) is an attractive therapeutic cytokine, with positive responses achieved in multiple OC clinical trials. However, clinical application of IFN-γ in OC is still hindered, due to the severe toxicity when used at higher levels, as well as the considerable pro-metastatic adverse effect when used at lower levels. Thus, an effective combined intervention is needed to enhance the anti-tumor efficacy of IFN-γ and to suppress the IFN-γ-induced metastasis. Here, we uncovered that OC cells develop an adaptive strategy by upregulating midkine (MDK) to counteract the IFN-γ-induced anti-tumor activity and to fuel IFN-γ-induced metastasis. We showed that MDK is a critical downstream target of IFN-γ in OC, and that this regulation acts in a dose-dependent manner and is mediated by STAT1. Gain-of-function studies showed that MDK overexpression promotes cell proliferation and metastasis in OC, indicating that IFN-γ-activated MDK may antagonize IFN-γ in inhibiting OC proliferation but synergize IFN-γ in promoting OC metastasis. Subsequently, we assessed the influence of MDK inhibition on IFN-γ-induced anti-proliferation and pro-metastasis effects using an MDK inhibitor (iMDK), and we found that MDK inhibition robustly enhanced IFN-γ-induced growth inhibition (all CIs < 0.1) and reversed IFN-γ-driven epithelial-to-mesenchymal transition (EMT) and metastasis in OC in vitro. Collectively, these data identify an IFN-γ responsive protein, MDK, in counteracting anti-proliferation while endowing the pro-metastatic role of IFN-γ in cancer treatment, and we therefore propose the combined utilization of the MDK inhibitor in IFN-γ-based therapies in future OC treatment.

Efficacy of immunotherapy is limited in patients with colorectal cancer (CRC) because high expression of tumor-derived transforming growth factor (TGF)-β pathway molecules and interferon (IFN)-stimulated genes (ISGs) promotes tumor immune evasion. Here, we identified a long noncoding RNA (lncRNA), VPS9D1-AS1, which was located in ribosomes and amplified TGF-β signaling and ISG expression. We show that high expression of VPS9D1-AS1 was negatively associated with T lymphocyte infiltration in two independent cohorts of CRC. VPS9D1-AS1 served as a scaffolding lncRNA by binding with ribosome protein S3 (RPS3) to increase the translation of TGF-β, TGFBR1, and SMAD1/5/9. VPS9D1-AS1 knockout downregulated OAS1, an ISG gene, which further reduced IFNAR1 levels in tumor cells. Conversely, tumor cells overexpressing VPS9D1-AS1 were resistant to CD8 + T cell killing and lowered IFNAR1 expression in CD8 + T cells. In a conditional overexpression mouse model, VPS9D1-AS1 enhanced tumorigenesis and suppressed the infiltration of CD8 + T cells. Treating tumor-bearing mice with antisense oligonucleotide drugs targeting VPS9D1-AS1 significantly suppressed tumor growth. Our findings indicate that the tumor-derived VPS9D1-AS1/TGF-β/ISG signaling cascade promotes tumor growth and enhances immune evasion and may thus serve as a potential therapeutic target for CRC., Competing Interests: LY, XD, ZL, JT, XH, TW, HQ, ZW No competing interests declared, (© 2022, Yang et al.)

Purpose. To determine the efficacy of electroacupuncture on recovering postanesthetic bladder function. Materials and Methods. Sixty-one patients undergoing spinal anaesthesia were recruited and allocated into electroacupuncture or control group randomly. Patients in electroacupuncture group received electroacupuncture therapy whereas ones in control group were not given any intervention. Primary endpoint was incidence of bladder overdistension and postoperative urinary retention. Secondary endpoints included time to spontaneous micturition, voided volume, and adverse events. Results. All patients (31 in electroacupuncture group and 30 in control group) completed the evaluation. During postoperative follow-up, patients in electroacupuncture group presented a significant lower proportion of bladder overdistension than counterparts in control group (16.1% versus 53.3%, P < 0.01). However, no significant difference was found in incidence of postoperative urinary retention between the two groups (0% versus 6.7%, P > 0.05). Furthermore, a shorter time to spontaneous micturition was found in electroacupuncture group compared to control group (228 min versus 313 min, P < 0.001), whereas urine volume and adverse events had no significant difference between the two groups. Conclusions. Electroacupuncture reduced the proportion of bladder overdistension and shortened the time to spontaneous micturition in patients undergoing spinal anesthesia. Electroacupuncture may be a therapeutic strategy for postanesthetic bladder dysfunction.