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BackgroundAs the selective inhibitor of BRAF kinase, vemurafenib exhibits effective antitumor activities in patients with V600 BRAF mutant melanomas. However, acquired drug resistance invariably develops after its initial treatment.MethodsImmunohistochemical staining was performed to detect the expression of iNOS and hTERT, p-p65, Epcam, CD44, PCNA in mice with melanoma xenografts. The proliferation and migration of melanoma cells were detected by MTT, tumorsphere culture, cell cycle, cell apoptosis, AO/EB assay and colony formation, transwell assay and scratch assay in vitro, and tumor growth differences were observed in xenograft nude mice. Changes in the expression of key molecules in the iNOS/hTERT signaling pathways were detected by western blot. Nucleus-cytoplasm separation, and immunofluorescence analyses were conducted to explore the location of p50/p65 in melanoma cell lines. Flow cytometry assay were performed to determine the expression of CD44. Pull down assay and ChIP assay were performed to detect the binding ability of p65 at iNOS and hTERT promoters. Additionally, hTERT promoter-driven luciferase plasmids were transfected in to melanoma cells with indicated treatment to determine luciferase activity of hTERT.ResultsMelatonin significantly and synergistically enhanced vemurafenib-mediated inhibitions of proliferation, colony formation, migration and invasion and promoted vemurafenib-induced apoptosis, cell cycle arresting and stemness weakening in melanoma cells. Further mechanism study revealed that melatonin enhanced the antitumor effect of vemurafenib by abrogating nucleus translocation of NF-κB p50/p65 and their binding at iNOS and hTERT promoters, thereby suppressing the expression of iNOS and hTERT. The elevated anti-tumor capacity of vemurafenib upon co-treatment with melatonin was also evaluated and confirmed in mice with melanoma xenografts.ConclusionsCollectively, our results demonstrate melatonin synergizes the antitumor effect of vemurafenib in human melanoma by inhibiting cell proliferation and cancer-stem cell traits via targeting NF-κB/iNOS/hTERT signaling pathway, and suggest the potential of melatonin in antagonizing the toxicity of vemurafenib and augmenting its sensitivities in melanoma treatment.

Background: Liver metastasis poses a significant barrier to effective immunotherapy in patients with colorectal cancer. Cryoablation has emerged as a vital supplementary therapeutic approach for these patients. However, its impact on the tumor microenvironment following the ablation of liver metastases remains unclear. Methods: We acquired multi-omics time-series data at 1 day, 5 days, and 14 days post-cryoablation, based on tumor and peripheral blood samples from clinical patients, cell co-culture models, and a liver metastases mouse model built on the MC38 cell line in C57BL/6 J mice. This dataset included single-cell transcriptomic sequencing, bulk tissue transcriptomic sequencing, 4D-Label-Free proteomics, flow cytometry data, western blot data, and histological immunofluorescence staining of pathological specimens. Results: We found that a neutrophil-related inflammatory state persisted for at least 14 days post-cryoablation. During this period, neutrophils underwent phenotypic changes, shifting from the N1 to the N2 type. Cryoablation also caused a significant increase in intracellular Ca2+ concentration in neutrophils, which triggered the formation of PAD4-dependent neutrophil extracellular traps (NETs), further promoting immune evasion. Moreover, animal studies demonstrated that depleting or inhibiting the CXCL2-CXCR2 signaling axis within neutrophils, or degrading NETs, could effectively restore the host's anti-tumor immune response. Conclusions: These findings underscore the critical role of neutrophils and their NETs in immune escape following cryoablation. Targeting the CXCL2-CXCR2-Ca2+-PAD4 axis could enhance the therapeutic response to PD-1 antibodies, providing a potential strategy to improve treatment outcomes for colorectal cancer with liver metastases. [ABSTRACT FROM AUTHOR]

BackgroundN-myc (and STAT) interactor (NMI) plays vital roles in tumor growth, progression, and metastasis. In this study, we identified NMI as a potential tumor suppressor in lung cancer and explored its molecular mechanism involved in lung cancer progression.MethodsHuman lung cancer cell lines and a mouse xenograft model was used to study the effect of NMI on tumor growth. The expression of NMI, COX-2 and relevant signaling proteins were examined by Western blot. Tissue microarray immunohistochemical analysis was performed to assess the correlation between NMI and COX-2 expression in lung cancer patients.ResultsNMI was highly expressed in normal lung cells and tissues, but lowly expressed in lung cancer cells and tissues. Overexpression of NMI induced apoptosis, suppressed lung cancer cell growth and migration, which were mediated by up-regulation of the cleaved caspase-3/9 and down-regulation of phosphorylated PI3K/AKT, MMP2/MMP9, β-cadherin, and COX-2/PGE2. In contrast, knockdown of NMI promoted lung cancer cell colony formation and migration, which were correlated with the increased expression of phosphorylated PI3K/AKT, MMP2/MMP9, β-cadherin and COX-2/PGE2. Further study showed that NMI suppressed COX-2 expression through inhibition of the p50/p65 NF-κB acetylation mediated by p300. The xenograft lung cancer mouse models also confirmed the NMI-mediated suppression of tumor growth by inhibiting COX-2 signaling. Moreover, tissue microarray immunohistochemical analysis of lung adenocarcinomas also demonstrated a negative correlation between NMI and COX-2 expression. Kaplan-Meier analysis indicated that the patients with high level of NMI had a significantly better prognosis.ConclusionsOur study showed that NMI suppressed tumor growth by inhibiting PI3K/AKT, MMP2/MMP9, COX-2/PGE2 signaling pathways and p300-mediated NF-κB acetylation, and predicted a favorable prognosis in human lung adenocarcinomas, suggesting that NMI was a potential tumor suppressor in lung cancer.

Melanoma is an aggressive cutaneous malignancy, illuminating the exact mechanisms and finding novel therapeutic targets are urgently needed. In this study, we identified KMT2A as a potential target, which promoted the growth of human melanoma cells. KMT2A knockdown significantly inhibited cell viability and cell migration and induced apoptosis, whereas KMT2A overexpression effectively promoted cell proliferation in various melanoma cell lines. Further study showed that KMT2A regulated melanoma cell growth by targeting the hTERT-dependent signal pathway. Knockdown of KMT2A markedly inhibited the promoter activity and expression of hTERT, and hTERT overexpression rescued the viability inhibition caused by KMT2A knockdown. Moreover, KMT2A knockdown suppressed tumorsphere formation and the expression of cancer stem cell markers, which was also reversed by hTERT overexpression. In addition, the results from a xenograft mouse model confirmed that KMT2A promoted melanoma growth via hTERT signaling. Finally, analyses of clinical samples demonstrated that the expression of KMT2A and hTERT were positively correlated in melanoma tumor tissues, and KMT2A high expression predicted poor prognosis in melanoma patients. Collectively, our results indicate that KMT2A promotes melanoma growth by activating the hTERT signaling, suggesting that the KMT2A/hTERT signaling pathway may be a potential therapeutic target for melanoma.

N-myc and STAT interactor (NMI) has been proved to bind to different transcription factors to regulate a variety of signaling mechanisms including DNA damage, cell cycle and epithelial-mesenchymal transition. However, the role of NMI in the regulation of cancer stem cells (CSCs) remains poorly understood. In this study, we investigated the regulation of NMI on CSCs traits in breast cancer and uncovered the underlying molecular mechanisms. We found that NMI was lowly expressed in breast cancer stem cells (BCSCs)-enriched populations. Knockdown of NMI promoted CSCs traits while its overexpression inhibited CSCs traits, including the expression of CSC-related markers, the number of CD44+CD24- cell populations and the ability of mammospheres formation. We also found that NMI-mediated regulation of BCSCs traits was at least partially realized through the modulation of hTERT signaling. NMI knockdown upregulated hTERT expression while its overexpression downregulated hTERT in breast cancer cells, and the changes in CSCs traits and cell invasion ability mediated by NMI were rescued by hTERT. The in vivo study also validated that NMI knockdown promoted breast cancer growth by upregulating hTERT signaling in a mouse model. Moreover, further analyses for the clinical samples demonstrated that NMI expression was negatively correlated with hTERT expression and the low NMI/high hTERT expression was associated with the worse status of clinical TNM stages in breast cancer patients. Furthermore, we demonstrated that the interaction of YY1 protein with NMI and its involvement in NMI-mediated transcriptional regulation of hTERT in breast cancer cells. Collectively, our results provide new insights into understanding the regulatory mechanism of CSCs and suggest that the NMI-YY1-hTERT signaling axis may be a potential therapeutic target for breast cancers.

Cyclooxygenase-2 (COX-2) plays an important role in lung cancer development and progression. Using streptavidin-agarose pulldown and proteomics assay, we identified and validated Ku80, a dimer of Ku participating in the repair of broken DNA double strands, as a new binding protein of the COX-2 gene promoter. Overexpression of Ku80 up-regulated COX-2 promoter activation and COX-2 expression in lung cancer cells. Silencing of Ku80 by siRNA down-regulated COX-2 expression and inhibited tumor cell growth in vitro and in a xenograft mouse model. Ku80 knockdown suppressed phosphorylation of ERK, resulting in an inactivation of the MAPK pathway. Moreover, CBP, a transcription co-activator, interacted with and acetylated Ku80 to co-regulate the activation of COX-2 promoter. Overexpression of CBP increased Ku80 acetylation, thereby promoting COX-2 expression and cell growth. Suppression of CBP by a CBP-specific inhibitor or siRNA inhibited COX-2 expression as well as tumor cell growth. Tissue microarray immunohistochemical analysis of lung adenocarcinomas revealed a strong positive correlation between levels of Ku80 and COX-2 and clinicopathologic variables. Overexpression of Ku80 was associated with poor prognosis in patients with lung cancers. We conclude that Ku80 promotes COX-2 expression and tumor growth and is a potential therapeutic target in lung cancer.

Melatonin is a hormone identified in plants and pineal glands of mammals and possesses diverse physiological functions. Fisetin is a bio-flavonoid widely found in plants and exerts antitumor activity in several types of human cancers. However, the combinational effect of melatonin and fisetin on antitumor activity, especially in melanoma treatment, remains unclear. Here, we tested the hypothesis that melatonin could enhance the antitumor activity of fisetin in melanoma cells and identified the underlying molecular mechanisms. The combinational treatment of melanoma cells with fisetin and melatonin significantly enhanced the inhibitions of cell viability, cell migration and clone formation, and the induction of apoptosis when compared with the treatment of fisetin alone. Moreover, such enhancement of antitumor effect by melatonin was found to be mediated through the modulation of the multiply signaling pathways in melanoma cells. The combinational treatment of fisetin with melatonin increased the cleavage of PARP proteins, triggered more release of cytochrome-c from the mitochondrial inter-membrane, enhanced the inhibition of COX-2 and iNOS expression, repressed the nuclear localization of p300 and NF-κB proteins, and abrogated the binding of NF-κB on COX-2 promoter. Thus, these results demonstrated that melatonin potentiated the anti-tumor effect of fisetin in melanoma cells by activating cytochrome-c-dependent apoptotic pathway and inhibiting COX-2/iNOS and NF-κB/p300 signaling pathways, and our study suggests the potential of such a combinational treatment of natural products in melanoma therapy.