TRPV1 inhibition suppresses non-small cell lung cancer progression by inhibiting tumour growth and enhancing the immune response.

PURPOSE: TRPV1 is a nonselective Ca2+ channel protein that is widely expressed and plays an important role during the occurrence and development of many cancers. Activation of TRPV1 channels can affect tumour progression by regulating proliferation, apoptosis and migration. Some studies have also shown that activating TRPV1 can affect tumour progression by modulating tumour immunity. However, the effects of TRPV1 on the development of non-small cell lung cancer (NSCLC) have not been explored clearly. METHOD: The Cancer Genome Atlas (TCGA) database and spatial transcriptomics datasets from 10 × Genomics were used to analyze TRPV1 expression in various tumour tissues. Cell proliferation and apoptosis were examined by cell counting kit 8 (CCK8), colony formation, and flow cytometry. Immunohistochemistry, qPCR, and western blotting were used to determine the mRNA and protein expression levels of TRPV1 and other related molecules. Tumour xenografts in BALB/C and C57BL/6J mice were used to determine the effects of TRPV1 on NSCLC development in vivo. Neurotransmitter content was examined by LC-MS/MS, ELISA and Immunohistochemistry. Immune cell infiltration was assessed by flow cytometry. RESULTS: In this study, we found that TRPV1 expression was significantly upregulated in NSCLC and that patients with high TRPV1 expression had a poor prognosis. TRPV1 knockdown can significantly inhibit NSCLC proliferation and induce cell apoptosis through Ca2+-IGF1R signaling. In addition, TRPV1 knockdown resulted in increased infiltration of CD4+ T cells, CD8+ T cells, GZMB+CD8+ T cells and DCs and decreased infiltration of immunosuppressive MDSCs in NSCLC. In addition, TRPV1 knockout effectively decreased the expression of M2 macrophage markers CD163 and increased the expression of M1-associated, costimulatory markers CD86. Knockdown or knockout of TRPV1 significantly inhibit tumour growth and promoted an antitumour immune response through supressing γ-aminobutyric acid (GABA) secretion in NSCLC. CONCLUSION: Our study suggests that TRPV1 acts as a tumour promoter in NSCLC, mediating pro-proliferative and anti-apoptotic effects on NSCLC through IGF1R signaling and regulating GABA release to affect the tumour immune response.

Molecular mechanism of XB130 adaptor protein mediates trastuzumab resistance in gastric cancer

Background Recent studies have shown that the activation of PI3K/AKT signaling pathway is an essential molecular mechanism participating in trastuzumab resistance in HER2 + GC (gastric cancer). However, how can we effectively inhibit AKT activity associated with drug resistance during trastuzumab treatment? Screening inhibitors against the upstream receptors of PI3K/AKT signaling pathway or interacting proteins of members has become an important way. Methods In this study, western blot, qRT-PCR, CCK8, Co-IP and other techniques were used to explore possible mechanisms participating in trastuzumab resistance in vitro. Besides, the xenograft mouse model and GC tissue samples from patients were used to further validate the in-vitro results. Results The expression of XB130 adaptor protein was remarkably increased in GC cell lines resistant to trastuzumab, and knockdown of XB130 could reverse the resistance via downregulating p-AKT. In addition, p-SRC (Tyr416) was increased in resistant cells, which could facilitate the binding of XB130 to PI3K p85α. It was also discovered that XB130 could negatively regulate PTEN gene transcription, and thus a positive feedback loop was formed between SRC-XB130-PTEN. Conclusions In HER2 + GC, XB130 contributes to trastuzumab resistance by stimulating the PI3K/AKT signaling pathway through binding to PI3K p85α under the mediation of SRC kinase and regulating PTEN gene transcription, and in turn forming a positive feedback loop between SRC-XB130-PTEN (AU)

The N6-methyladenosine METTL3 regulates tumorigenesis and glycolysis by mediating m6A methylation of the tumor suppressor LATS1 in breast cancer.

BACKGROUND: Posttranscriptional modification of tumor-associated factors plays a pivotal role in breast cancer progression. However, the underlying mechanism remains unknown. M6A modifications in cancer cells are dynamic and reversible and have been found to impact tumor initiation and progression through various mechanisms. In this study, we explored the regulatory mechanism of breast cancer cell proliferation and metabolism through m6A methylation in the Hippo pathway.  METHODS: A combination of MeRIP-seq, RNA-seq and metabolomics-seq was utilized to reveal a map of m6A modifications in breast cancer tissues and cells. We conducted RNA pull-down assays, RIP-qPCR, MeRIP-qPCR, and RNA stability analysis to identify the relationship between m6A proteins and LATS1 in m6A regulation in breast cancer cells. The expression and biological functions of m6A proteins were confirmed in breast cancer cells in vitro and in vivo. Furthermore, we investigated the phosphorylation levels and localization of YAP/TAZ to reveal that the activity of the Hippo pathway was affected by m6A regulation of LATS1 in breast cancer cells.  RESULTS: We demonstrated that m6A regulation plays an important role in proliferation and glycolytic metabolism in breast cancer through the Hippo pathway factor, LATS1. METTL3 was identified as the m6A writer, with YTHDF2 as the reader protein of LATS1 mRNA, which plays a positive role in promoting both tumorigenesis and glycolysis in breast cancer. High levels of m6A modification were induced by METTL3 in LATS1 mRNA. YTHDF2 identified m6A sites in LATS1 mRNA and reduced its stability. Knockout of the protein expression of METTL3 or YTHDF2 increased the expression of LATS1 mRNA and suppressed breast cancer tumorigenesis by activating YAP/TAZ in the Hippo pathway. CONCLUSIONS: In summary, we discovered that the METTL3-LATS1-YTHDF2 pathway plays an important role in the progression of breast cancer by activating YAP/TAZ in the Hippo pathway.

Molecular mechanism of XB130 adaptor protein mediates trastuzumab resistance in gastric cancer.

BACKGROUND: Recent studies have shown that the activation of PI3K/AKT signaling pathway is an essential molecular mechanism participating in trastuzumab resistance in HER2 + GC (gastric cancer). However, how can we effectively inhibit AKT activity associated with drug resistance during trastuzumab treatment? Screening inhibitors against the upstream receptors of PI3K/AKT signaling pathway or interacting proteins of members has become an important way. METHODS: In this study, western blot, qRT-PCR, CCK8, Co-IP and other techniques were used to explore possible mechanisms participating in trastuzumab resistance in vitro. Besides, the xenograft mouse model and GC tissue samples from patients were used to further validate the in-vitro results. RESULTS: The expression of XB130 adaptor protein was remarkably increased in GC cell lines resistant to trastuzumab, and knockdown of XB130 could reverse the resistance via downregulating p-AKT. In addition, p-SRC (Tyr416) was increased in resistant cells, which could facilitate the binding of XB130 to PI3K p85α. It was also discovered that XB130 could negatively regulate PTEN gene transcription, and thus a positive feedback loop was formed between SRC-XB130-PTEN. CONCLUSIONS: In HER2 + GC, XB130 contributes to trastuzumab resistance by stimulating the PI3K/AKT signaling pathway through binding to PI3K p85α under the mediation of SRC kinase and regulating PTEN gene transcription, and in turn forming a positive feedback loop between SRC-XB130-PTEN.

Ozone induces BEL7402 cell apoptosis by increasing reactive oxygen species production and activating JNK.

BACKGROUND: Oxidative stress is an important factor in the modulation of both tumorigenesis and anticancer responses. Ozone (O3) is a strong oxidant that causes redox reactions and exerts anticancer effects in various types of cancer cells. However, the pathways involved in O3-induced cell death are not well understood. METHODS: In vitro human hepatocellular carcinoma (HCC) BEL7402 cells were treated with various O3 concentrations to evaluate O3 cytotoxicity by Cell Counting Kit-8 (CCK-8) assay and flow cytometry. The regulatory mechanisms were analyzed by western blot analysis. In vivo, an HCC model was established to evaluate the inhibition of HCC with O3 treatment. RESULTS: In vitro cells treated with O3 exhibited a round and small morphology with nuclear shrinkage and fragmentation. The CCK-8 assay confirmed the potent cytotoxic activity of O3 against BEL7402 cells (IC50 value of 5 µg/mL). Acridine orange/ethidium bromide (AO/EB) staining revealed apoptosis of BEL7402 cells after O3 treatment. Flow cytometry analysis showed that S phase cell cycle arrest and apoptosis increased with O3 exposure. In addition, O3 exposure reduced the mitochondrial membrane potential (ΔΨm) and induced reactive oxygen species (ROS) accumulation. Western blot analysis showed that O3 exposure reduced B-cell lymphoma 2 (BCL-2) expression and increased cleaved poly ADP-ribose polymerase (PARP), cytochrome C (Cyt-C), caspase-3, caspase-9, and p-JNK expression. In vivo, treatment with intratumor injection O3 (20 µg/mL) inhibited HCC growth. CONCLUSIONS: Overall, our findings showed that O3 induces BEL7402 cell apoptosis via the intrinsic mitochondria-dependent pathway. Therefore, O3 has therapeutic potential for HCC.

miR-301a-3p induced by endoplasmic reticulum stress mediates the occurrence and transmission of trastuzumab resistance in HER2-positive gastric cancer.

Trastuzumab resistance negatively influences the clinical efficacy of the therapy for human epidermal growth factor receptor 2 (HER2) positive gastric cancer (GC), and the underlying mechanisms remain elusive. Exploring the mechanisms and finding effective approaches to address trastuzumab resistance are of great necessity. Here, we confirmed that endoplasmic reticulum (ER) stress-induced trastuzumab resistance by up-regulating miR-301a-3p in HER2-positive GC cells. Moreover, we elucidated that miR-301a-3p mediated trastuzumab resistance by down-regulating the expression of leucine-rich repeats and immunoglobulin-like domains containing protein 1 (LRIG1) and subsequently activating the expression of insulin-like growth factor 1 receptor (IGF-1R) and fibroblast growth factor receptor 1 (FGFR1) under ER stress. We also found that intercellular transfer of miR-301a-3p by exosomes disseminated trastuzumab resistance. The present study demonstrated that exosomal miR-301a-3p could serve as a non-invasive biomarker for trastuzumab resistance, which was maybe a novel potential therapeutic target to overcome trastuzumab resistance and improve the curative effect of trastuzumab in HER2-positive GC patients.

Kin17 knockdown suppresses the migration and invasion of cervical cancer cells through NF-κB-Snail pathway.

Cervical cancer is one of the most common cancers in women worldwide. Metastasis in cancer has been a Gordian knot due to unsatisfactory clinical treatments. KIN17, a highly conserved gene from yeast to human, up-regulation is associated with the pathogenesis and development of several common cancers. Our previous works revealed that elevated expression of kin17 observed in cervical cancer tissues showed a close association with lymph node metastasis. This study aimed to explore roles and mechanisms of kin17 in the migration and invasion of cervical cancer cells. Cervical cancer cell lines HeLa and SiHa with kin17 knockdown were constructed by using recombinant lentiviral vector that carry specific siRNA targeting KIN17 gene. The mRNA and protein levels of kin17 in cells were determined by RT-qPCR and western blotting, respectively. Wound healing assay and transwell assays were performed to assess the migration and invasion abilities of the cancer cells, respectively. The expression of signaling proteins involved in the NF-κB-Snail pathway was analyzed by western blotting. As our results showed, the mRNA and protein levels of kin17 in HeLa cells and SiHa cells showed a significant decrease by transfection with recombinant lentiviral vector carrying specific siRNA. Compared with control group, the migration rates were decreased in the kin17 knockdown group in both HeLa and SiHa cell lines in wound healing assay as well as transwell assay without matrigel. Kin17 knockdown also reduced the cell invasion number of both HeLa and SiHa cells. In addition, the phosphorylation of nuclear factor Kαppa B (NF-κB) p65, IKαppa B kinase α (IKKα), and IKαppa B α (IκBα) in NF-κB pathway and the expression of Snail were decreased in HeLa cells and SiHa cells by kin17 knockdown. Our results demonstrated that knockdown of kin17 in cervical cancer cells suppressed cell migration and invasion, and inhibited the activity of NF-κB signaling pathway and the expression of Snail. These findings suggested kin17 as an essential regulator of the cell migration and invasion and the underlying molecular mechanism involved NF-κB-Snail pathway in cervical cancer. This might serve as a novel molecular therapeutic target for treating cervical cancer metastasis.