PHLDA2 overexpression facilitates senescence and apoptosis via the mitochondrial route in human nucleus pulposus cells by regulating Wnt/ß-catenin signalling pathway.

Low back pain is a common clinical symptom of intervertebral disc degeneration (IVDD), which seriously affects the quality of life of the patients. The abnormal apoptosis and senescence of nucleus pulposus cells (NPCs) play important roles in the pathogenesis of IVDD. PHLDA2 is an imprinted gene related to cell apoptosis and tumour progression. However, its role in NPC degeneration is not yet clear. Therefore, this study was set to explore the effects of PHLDA2 on NPC senescence and apoptosis and the underlying mechanisms. The expression of PHLDA2 was examined in human nucleus pulposus (NP) tissues and NPCs. Immunohistochemical staining, magnetic resonance imaging imaging and western blot were performed to evaluate the phenotypes of intervertebral discs. Senescence and apoptosis of NPCs were assessed by SA-ß-galactosidase, flow cytometry and western blot. Mitochondrial function was investigated by JC-1 staining and transmission electron microscopy. It was found that the expression level of PHLDA2 was abnormally elevated in degenerated human NP tissues and NPCs. Furthermore, knockdown of PHLDA2 can significantly inhibit senescence and apoptosis of NPCs, whereas overexpression of PHLDA2 can reverse senescence and apoptosis of NPCs in vitro. In vivo experiment further confirmed that PHLDA2 knockdown could alleviate IVDD in rats. Knockdown of PHLDA2 could also reverse senescence and apoptosis in IL-1ß-treated NPCs. JC-1 staining indicated PHLDA2's knockdown impaired disruption of the mitochondrial membrane potential and also ameliorated superstructural destruction of NPCs as showed by transmission electron microscopy. Finally, we found the PHLDA2 knockdown promoted Collagen-II expression and suppressed MMP3 expression in NPCs by repressing wnt/ß-catenin pathway. In conclusion, the results of the present study showed that PHLDA2 promotes IL-1ß-induced apoptosis and senescence of NP cells via mitochondrial route by activating the Wnt/ß-catenin pathway, and suggested that therapy targeting PHLDA2 may provide valuable insights into possible IVDD therapies.

TC2N inhibits distant metastasis and stemness of breast cancer via blocking fatty acid synthesis.

BACKGROUND: Tandem C2 domains, nuclear (TC2N) is a C2 domain-containing protein that belongs to the carboxyl-terminal type (C-type) tandem C2 protein family, and acts as an oncogenic driver in several cancers. Previously, we preliminarily reported that TC2N mediates the PI3K-Akt signaling pathway to inhibit tumor growth of breast cancer (BC) cells. Beyond that, its precise biological functions and detailed molecular mechanisms in BC development and progression are not fully understood. METHODS: Tumor tissues of 212 BC patients were subjected to tissue microarray and further assessed the associations of TC2N expression with pathological parameters and FASN expression. The protein levels of TC2N and FASN in cell lines and tumor specimens were monitored by qRT-PCR, WB, immunofluorescence and immunohistochemistry. In vitro cell assays, in vivo nude mice model was used to assess the effect of TC2N ectopic expression on tumor metastasis and stemness of breast cancer cells. The downstream signaling pathway or target molecule of TC2N was mined using a combination of transcriptomics, proteomics and lipidomics, and the underlying mechanism was explored by WB and co-IP assays. RESULTS: Here, we found that the expression of TC2N remarkedly silenced in metastatic and poorly differentiated tumors. Function-wide, TC2N strongly inhibits tumor metastasis and stem-like properties of BC via inhibition of fatty acid synthesis. Mechanism-wise, TC2N blocks neddylated PTEN-mediated FASN stabilization by a dual mechanism. The C2B domain is crucial for nuclear localization of TC2N, further consolidating the TRIM21-mediated ubiquitylation and degradation of FASN by competing with neddylated PTEN for binding to FASN in nucleus. On the other hand, cytoplasmic TC2N interacts with import proteins, thereby restraining nuclear import of PTEN to decrease neddylated PTEN level. CONCLUSIONS: Altogether, we demonstrate a previously unidentified role and mechanism of TC2N in regulation of lipid metabolism and PTEN neddylation, providing a potential therapeutic target for anti-cancer.

Long noncoding RNA XLOC_006786 inhibits the proliferation, invasion and metastasis of osteosarcoma cells through NOTCH3 signaling pathway by targeting miR-491-5p.

Recent research has indicated that Long noncoding RNAs (LncRNAs) are crucial in many disorders, especially tumors. However, the exact role of LncRNA XLOC_006786 (LncRNA-SPIDR-2:1) in malignancies, especially in human osteosarcoma, is unclear. The results of RT‒qPCR, western blotting, CCK-8 assays, and Transwell assays showed that LncRNA XLOC_006786 inhibited osteosarcoma cell proliferation, invasion, and migration, indicating that it may be a tumor suppressor gene in osteosarcoma. We found that LncRNA XLOC_006786 negatively regulated NOTCH3, which is an oncogenic gene in osteosarcoma, as we previously reported. Bioinformatics analysis showed that miR-491-5p may be a direct target of LncRNA XLOC_006786, while NOTCH3 is a key target of miR-491-5p. Then, we verified that LncRNA XLOC_006786 could prevent lung metastatic osteosarcoma in vivo. Taken together, our research showed that LncRNA XLOC_006786 suppresses osteosarcoma proliferation, invasion, and metastasis through the NOTCH3 signaling pathway by targeting miR-491-5p.

Gene Expression Profile and Prognostic Value of m6A RNA Methylation Regulators in Hepatocellular Carcinoma.

BACKGROUND: N6-methyladenosine (m6A) RNA methylation is the most prevalent modification of mammalian RNA, and it is associated with tumorigenesis and cancer progression. Its regulation is mediated via m6A-related regulators, including "erasers," "readers," and "writers". The present study evaluated the expression profile, risk signature and prognostic value of 13 m6A regulators in hepatocellular carcinoma (HCC) using different datasets, including The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO) and clinical samples. METHODS: We used 374 HCC samples derived from the TCGA database, 569 HCC samples from 2 GEO datasets, and clinical tumour and nontumour tissues derived from 60 patients with HCC who underwent surgery in Xinqiao Hospital Chongqing to assess the gene expression profiles and prognostic values of m6A-related regulators in HCC. RESULTS: Eight of 13 core m6A-related regulators were overexpressed in all databases, including TCGA, GSE, clinical tumour and nontumour tissues of HCC. Two clusters (Cluster 1 and Cluster 2) were identified via consensus clustering. Cluster 2 was associated with poorer prognosis, higher tumour grade, higher AFP levels, and worse outcome compared to Cluster 1, which indicates that these m6A-related regulators are highly correlated with HCC malignancy. We performed survival analyses using the Log rank tests and a Cox regression model. Gene enrichment analysis was used to detect the related KEGG and GO pathways. We derived a prognostic risk signature using five selected m6A-related regulators. CONCLUSION: Our work suggested that m6A-related regulators might be key participants in the tumour progression of HCC and potential biomarkers with prognostic value.

Overexpression of chemokine receptor lymphotactin receptor 1 has prognostic value in clear cell renal cell carcinoma.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is an aggressive subtype of renal cell carcinoma. X-C motif chemokine receptor 1 (XCR1) exerts important roles in tumor progression; however, its role in ccRCC is unclear. METHODS: We utilized publicly available data from The Cancer Genome Atlas (TCGA) to assess the role of XCR1 in ccRCC and validated the results in 36 samples from patients with ccRCC who underwent curative resection in Xinqiao Hospital Chongqing. XCR1 overexpression was identified in ccRCC, which was confirmed by qRT-PCR assay and immunohistochemical staining of ccRCC samples. RESULTS: For the TCGA and clinical data, Kaplan-Meier survival analysis revealed that higher XCR1 expression in ccRCC was related to longer overall survival. Cox regression analysis suggested that XCR1 is an independent risk factor for ccRCC. GSEA analysis suggested that XCR1 is associated with the JAK/STAT signaling pathway. XCR1 knockdown by small interfering RNA (siRNA) significantly increased ccRCC cell proliferation and migration, and decreased cell apoptosis. CONCLUSION: We found higher XCR1 expression in ccRCC compared with that in normal tissues is related to longer overall survival in patients with ccRCC. XCR1 knockdown significantly increased RCC cells proliferation and migration, and decreased apoptosis. XCR1 might be used as a prognostic biomarker in ccRCC in the future.

High expression of ID1 facilitates metastasis in human osteosarcoma by regulating the sensitivity of anoikis via PI3K/AKT depended suppression of the intrinsic apoptotic signaling pathway.

A lack of understanding of the molecular basis underlying the regulation of metastatic disease and its effective therapy are the primary causes of high mortality in osteosarcoma. Thus, new insights into metastases and novel effective targets for metastatic osteosarcoma are urgently required. Anoikis resistance is considered a hallmark of cancer cells with metastatic ability. However, the molecular mechanism of anoikis is poorly understood in osteosarcoma. We applied immunohistochemistry to investigate the correlation between inhibitor of differentiation or DNA binding 1 (ID1) and clinicopathological features, and investigated the correlation between ID1 and the metastatic behavior of osteosarcoma cells, in vitro and in vivo. The results revealed that ID1 is overexpressed in human osteosarcoma tissues, is positively associated with lung metastases, and is a potential biomarker of poor prognosis. Overexpression of ID1 could increase anoikis insensitivity of osteosarcoma cells to facilitate metastasis through the PI3K/AKT-dependent mitochondrial apoptosis pathway. Knockdown of ID1 partly reversed the high potential of metastasis in anoikis-resistant osteosarcoma cells. Our findings revealed, that ID1 is a candidate molecular target for metastatic potential osteosarcoma by highlighting the role of anoikis resistance. In addition ID1 might be a potential predictor of poor prognosis in patients with osteosarcoma.

Overexpression of Notch3 is associated with metastasis and poor prognosis in osteosarcoma patients.

BACKGROUND: Notch signaling abnormalities are associated with the development of various tumors, including hematopoietic and epithelium-derived tumors. However, the role of Notch signaling in tumors originating from mesenchymal cells is unclear. The effect of Notch3 expression on the prognosis of osteosarcoma and its role and mechanism in osteosarcoma cells have never been reported. MATERIALS AND METHODS: In this study, we performed a clinicopathological analysis of 70 cases of osteosarcoma, with primary focus on survival. Osteosarcoma cell lines MTH and U2OS were used. After knockdown of Notch3 by lentiviral transfection and siRNA, the cell cycle, cell viability, and wound healing capacity were assessed. Subsequently, the Transwell assay was performed, and the expression levels of hairy and enhancer of split-1 (Hes1) and matrix metalloproteinase 7 (MMP7) were detected by RT-PCR and Western blot assay. The expression of MMP7 was also detected after knockdown of Hes1. Animal experiments were performed by injecting the cell lines MTH of Notch3 knockdown into mice tail veins and comparing the development of lung metastasis with the control group. RESULTS: Comparison of survival curves showed that Notch3 expression significantly impacts patient survival. Additionally, multivariate analysis revealed that Notch3 is an independent prognostic factor for osteosarcoma. In in vivo experiments, osteosarcoma-associated pulmonary metastasis in nude mice was reduced after Notch3 silencing. The expression of downstream effector molecule, Hes1, and that of the invasion and metastasis-associated proteolytic enzyme, MMP7, were reduced, and MMP7 was further decreased by Hes1 knockdown in in vitro experiments. CONCLUSION: Notch3 is a prognostic factor for osteosarcoma and might regulate its invasion and metastasis through the downstream target gene Hes1 and effector MMP7.

TSSC3 promotes autophagy via inactivating the Src-mediated PI3K/Akt/mTOR pathway to suppress tumorigenesis and metastasis in osteosarcoma, and predicts a favorable prognosis.

BACKGROUND: Over the last two or three decades, the pace of development of treatments for osteosarcoma tends has been slow. Novel effective therapies for osteosarcoma are still lacking. Previously, we reported that tumor-suppressing STF cDNA 3 (TSSC3) functions as an imprinted tumor suppressor gene in osteosarcoma; however, the underlying mechanism by which TSSC3 suppresses the tumorigenesis and metastasis remain unclear. METHODS: We investigated the dynamic expression patterns of TSSC3 and autophagy-related proteins (autophagy related 5 (ATG5) and P62) in 33 human benign bone tumors and 58 osteosarcoma tissues using immunohistochemistry. We further investigated the correlations between TSSC3 and autophagy in osteosarcoma using western blotting and transmission electronic microscopy. CCK-8, Edu, and clone formation assays; wound healing and Transwell assays; PCR; immunohistochemistry; immunofluorescence; and western blotting were used to investigated the responses in TSSC3-overexpressing osteosarcoma cell lines, and in xenografts and metastasis in vivo models, with or without autophagy deficiency caused by chloroquine or ATG5 silencing. RESULTS: We found that ATG5 expression correlated positively with TSSC3 expression in human osteosarcoma tissues. We demonstrated that TSSC3 was an independent prognostic marker for overall survival in osteosarcoma, and positive ATG5 expression associated with positive TSSC3 expression suggested a favorable prognosis for patients. Then, we showed that TSSC3 overexpression enhanced autophagy via inactivating the Src-mediated PI3K/Akt/mTOR pathway in osteosarcoma. Further results suggested autophagy contributed to TSSC3-induced suppression of tumorigenesis and metastasis in osteosarcoma in vitro and in vivo models. CONCLUSIONS: Our findings highlighted, for the first time, the importance of autophagy as an underlying mechanism in TSSC3-induced antitumor effects in osteosarcoma. We also revealed that TSSC3-associated positive ATG5 expression might be a potential predictor of favorable prognosis in patients with osteosarcoma.

TSSC3 represses self-renewal of osteosarcoma stem cells and Nanog expression by inhibiting the Src/Akt pathway.

Osteosarcoma is the most common type of bone cancer, and the second leading cause of cancer-related death in children and young adults. Osteosarcoma stem cells are essential for osteosarcoma initiation, metastasis, chemoresistance and recurrence. In the present study, we report that: 1) higher TSSC3 expression indicates a better prognosis for osteosarcoma patients, and; 2) overexpression of TSSC3 significantly decreases sphere-forming capacity, tumor initiation, stemness-related surface markers and Nanog expression in osteosarcoma cells. We also discovered that higher Nanog expression correlates to a worse prognosis for osteosarcoma patients, and overexpression of Nanog increases the stem-related phenotype in osteosarcoma cells. Knockdown of Nanog suppresses these phenotypes. Inhibition of Nanog expression and self-renewal of osteosarcoma cells by TSSC3 overexpression appears to be mediated through inactivation of the Src/Akt pathway. In the clinical setting, expression of TSSC3, p-Src and Nanog is associated with recurrence, metastasis and surgical intervention. Lower TSSC3 expression, higher Nanog expression or higher p-Src expression indicate a poor prognosis for osteosarcoma patients. Overall, our study demonstrates that TSSC3 inhibits the stem-like phenotype and Nanog expression by inactivation of the Src/Akt pathway; this emphasizes the importance of Nanog in osteosarcoma stem cells.

RanBP9/TSSC3 complex cooperates to suppress anoikis resistance and metastasis via inhibiting Src-mediated Akt signaling in osteosarcoma.

Suppression of anoikis is a prerequisite for tumor cell metastasis, which is correlated with chemoresistance and poor prognosis. We characterized a novel interaction between RanBP9 SPRY domain and TSSC3 PH domain by which RanBP9/TSSC3 complex exerts transcription and post-translation regulation in osteosarcoma. RanBP9/TSSC3 complex was inversely correlated with a highly anoikis-resistant phenotype in osteosarcoma cells and metastasis in human osteosarcoma. RanBP9 cooperated with TSSC3 to inhibit anchorage-independent growth and to promote anoikis in vitro and suppress lung metastasis in vivo. Moreover, RanBP9 SPRY domain was required for RanBP9/TSSC3 complex-mediated anoikis resistance. Mechanistically, RanBP9 formed a ternary complex with TSSC3 and Src to scaffold this interaction, which suppressed both Src and Src-dependent Akt pathway activations and facilitated mitochondrial-associated anoikis. Collectively, the newly identified RanBP9/TSSC3 complex cooperatively suppress metastasis via downregulation of Src-dependent Akt pathway to expedite mitochondrial-associated anoikis. This study provides a biological basis for exploring the therapeutic significance of dual targeting of RanBP9 and TSSC3 in osteosarcoma.