Targeting the mitochondrial protein YME1L to inhibit osteosarcoma cell growth in vitro and in vivo.

Exploring novel diagnostic and therapeutic biomarkers is extremely important for osteosarcoma. YME1 Like 1 ATPase (YME1L), locating in the mitochondrial inner membrane, is key in regulating mitochondrial plasticity and metabolic activity. Its expression and potential functions in osteosarcoma are studied in the present study. We show that YME1L mRNA and protein expression is significantly elevated in osteosarcoma tissues derived from different human patients. Moreover, its expression is upregulated in various primary and immortalized osteosarcoma cells. The Cancer Genome Atlas database results revealed that YME1L overexpression was correlated with poor overall survival and poor disease-specific survival in sarcoma patients. In primary and immortalized osteosarcoma cells, silencing of YME1L through lentiviral shRNA robustly inhibited cell viability, proliferation, and migration. Moreover, cell cycle arrest and apoptosis were detected in YME1L-silenced osteosarcoma cells. YME1L silencing impaired mitochondrial functions in osteosarcoma cells, causing mitochondrial depolarization, oxidative injury, lipid peroxidation and DNA damage as well as mitochondrial respiratory chain complex I activity inhibition and ATP depletion. Contrarily, forced YME1L overexpression exerted pro-cancerous activity and strengthened primary osteosarcoma cell proliferation and migration. YME1L is important for Akt-S6K activation in osteosarcoma cells. Phosphorylation of Akt and S6K was inhibited after YME1L silencing in primary osteosarcoma cells, but was strengthened with YME1L overexpression. Restoring Akt-mTOR activation by S473D constitutively active Akt1 mitigated YME1L shRNA-induced anti-osteosarcoma cell activity. Lastly, intratumoral injection of YME1L shRNA adeno-associated virus inhibited subcutaneous osteosarcoma xenograft growth in nude mice. YME1L depletion, mitochondrial dysfunction, oxidative injury, Akt-S6K inactivation, and apoptosis were detected in YME1L shRNA-treated osteosarcoma xenografts. Together, overexpressed YME1L promotes osteosarcoma cell growth, possibly by maintaining mitochondrial function and Akt-mTOR activation.

Crosstalk between m6A modification and alternative splicing during cancer progression.

Background N6-methyladenosine (m6A), the most prevalent internal mRNA modification in eukaryotes, is added by m6A methyltransferases, removed by m6A demethylases and recognised by m6A-binding proteins. This modification significantly influences carious facets of RNA metabolism and plays a pivotal role in cellular and physiological processes. Main body Pre-mRNA alternative splicing, a process that generates multiple splice isoforms from multi-exon genes, contributes significantly to the protein diversity in mammals. Moreover, the presence of crosstalk between m6A modification and alternative splicing, with m6A modifications on pre-mRNAs exerting regulatory control, has been established. The m6A modification modulates alternative splicing patterns by recruiting specific RNA-binding proteins (RBPs) that regulate alternative splicing or by directly influencing the interaction between RBPs and their target RNAs. Conversely, alternative splicing can impact the deposition or recognition of m6A modification on mRNAs. The integration of m6A modifications has expanded the scope of therapeutic strategies for cancer treatment, while alternative splicing offers novel insights into the mechanistic role of m6A methylation in cancer initiation and progression. Conclusion This review aims to highlight the biological functions of alternative splicing of m6A modification machinery and its implications in tumourigenesis. Furthermore, we discuss the clinical relevance of understanding m6A-dependent alternative splicing in tumour therapies.

Sorafenib induces ferroptosis by promoting TRIM54-mediated FSP1 ubiquitination and degradation in hepatocellular carcinoma.

BACKGROUND: Ferroptosis is a unique form of regulated cell death that provided a new opportunity for cancer therapy. Ferroptosis suppressor protein 1 (FSP1) is a key regulator in the NAD(P)H/FSP1/CoQ10 antioxidant system, which sever as an oxide redox enzyme to scavenge harmful lipid hydroperoxides and escape from ferroptosis in cells. This study aimed to investigate the role of FSP1 on sorafenib-induced ferroptosis and disclosed the underlying mechanisms. METHODS: Cell viability, malondialdehyde (MDA), glutathione (GSH), and lipid reactive oxygen species levels were assessed using indicated assay kits. The levels of FSP1 and glutathione peroxidase 4 (GPX4) in the patients with HCC were analyzed based on the database. Western blot and quantitative real-time PCR were performed to detect the protein and mRNA expression. Co-immunoprecipitation was applied to detect the interaction between proteins. Tumor xenograft experiments were used to evaluate whether overexpression of FSP1-inhibited sorafenib-induced ferroptosis in vivo. RESULTS: We verified that sorafenib-induced ferroptosis in HCC. Furthermore, we found that sorafenib decreased the protein level of FSP1, and knockdown FSP1 rendered HCC cells susceptible to sorafenib-induced ferroptosis. Co-immunoprecipitation and ubiquitination assays showed that sorafenib accelerated the TRIM54-mediated FSP1 ubiquitination and degradation. Sorafenib-induced ferroptosis was abrogated by TRIM54 suppression. Mechanically, sorafenib-promoted TRIM54 ubiquitinated and degraded FSP1 by means of the ERK pathway. Moreover, FSP1 enhanced tumor development and decreased HCC cellular susceptibility to sorafenib in vivo. CONCLUSIONS: Sorafenib facilitated the TRIM54-mediated FSP1 ubiquitination through the ERK pathway, thereby inducing ferroptosis in HCC cells.

HPV E7-drived ALKBH5 promotes cervical cancer progression by modulating m6A modification of PAK5.

Human papillomavirus (HPV) infection is a causative agent of cervical cancer (CC). N6-methyladenosine (m6A) modification is implicated in carcinogenesis and tumor progression. However, the involvement of m6A modification in HPV-involved CC remains unclear. Here we showed that HPV E6/7 oncoproteins affected the global m6A modification and E7 specifically promoted the expression of ALKBH5. We found that ALKBH5 was significantly upregulated in CC and might serve as a valuable prognostic marker. Forced expression of ALKBH5 enhanced the malignant phenotypes of CC cells. Mechanistically, we discovered that E7 increased ALKBH5 expression through E2F1-mediated activation of the H3K27Ac and H3K4Me3 histone modifications, as well as post-translational modification mediated by DDX3. ALKBH5-mediated m6A demethylation enhanced the expression of PAK5. The m6A reader YTHDF2 bound to PAK5 mRNA and regulated its stability in an m6A-dependent manner. Moreover, ALKBH5 promoted tumorigenesis and metastasis of CC by regulating PAK5. Overall, our findings herein demonstrate a significant role of ALKBH5 in CC progression in HPV-positive cells. Thus, we propose that ALKBH5 may serve as a prognostic biomarker and therapeutic target for CC patients.

PAK5 potentiates slug transactivation of N-cadherin to facilitate metastasis of renal cell carcinoma.

Renal cell carcinoma (RCC) is an aggravating cancer with a poor prognosis and a high rate of metastasis. PAK5, a p21-activated kinases, has shown to be overexpressed in a variety of cancers, including RCC. In previous studies, we discovered that PAK5 regulates cell migration and invasion in RCC cell lines. However, the underlying mechanisms remain obscure. In this study, we consolidated that PAK5 confers a pro-metastatic phenotype RCC cells in vitro and exacerbates metastasis in vivo. High PAK5 expression was associated with an advanced TNM stage and a lower overall survival. Furthermore, PAK5 increases the expression level of N-cadherin. In terms of mechanism, PAK5 bound to Slug and phosphorylated it at serine 87. As a result, phosphorylated Slug transactivated N-cadherin, accelerating the epithelial-mesenchymal transition. Collectively, Slug is a novel PAK5 substrate, and PAK5-mediated phosphorylation of Slug-S87 increases N-cadherin and the pro-metastatic phenotype of RCC, implying that phosphorylated Slug-S87 could be a therapeutic target in progressive RCC.

COPS6 promotes tumor progression and reduces CD8+ T cell infiltration by repressing IL-6 production to facilitate tumor immune evasion in breast cancer.

Due to poor T cell infiltration, tumors evade immune surveillance. Increased CD8+ T cell infiltration in breast cancer suggests a satisfactory response to immunotherapy. COPS6 has been identified as an oncogene, but its role in regulating antitumor immune responses has not been defined. In this study, we investigated the impact of COPS6 on tumor immune evasion in vivo. Tumor transplantation models were established in C57BL/6 J mice and BALB/c nude mice. Flow cytometry was conducted to identify the role of COPS6 on tumor-infiltrating CD8+ T cells. By analyzing the TCGA and GTEx cohort, we found that COPS6 expression was significantly up-regulated in a variety of cancers. In human osteosarcoma cell line U2OS and non-small cell lung cancer cell line H1299, we showed that p53 negatively regulated COPS6 promoter activity. In human breast cancer MCF-7 cells, COPS6 overexpression stimulated p-AKT expression as well as the proliferation and malignant transformation of tumor cells, whereas knockdown of COPS6 caused opposite effects. Knockdown of COPS6 also significantly suppressed the growth of mouse mammary cancer EMT6 xenografts in BALB/c nude mice. Bioinformatics analysis suggested that COPS6 was a mediator of IL-6 production in the tumor microenvironment and a negative regulator of CD8+ T cell tumor infiltration in breast cancer. In C57BL6 mice bearing EMT6 xenografts, COPS6 knockdown in the EMT6 cells increased the number of tumor-infiltrating CD8+ T cells, while knockdown of IL-6 in COPS6KD EMT6 cells diminished tumor infiltrating CD8+ T cells. We conclude that COPS6 promotes breast cancer progression by reducing CD8+ T cell infiltration and function via the regulation of IL-6 secretion. This study clarifies the role of p53/COPS6/IL-6/CD8+ tumor infiltrating lymphocytes signaling in breast cancer progression and immune evasion, opening a new path for development of COPS6-targeting therapies to enhance tumor immunogenicity and treat immunologically "cold" breast cancer.

NRP1 contributes to stemness and potentiates radioresistance via WTAP-mediated m6A methylation of Bcl-2 mRNA in breast cancer.

NRP1 is a transmembrane glycoprotein that is highly expressed in a variety of tumors. There is evidence that NRP1 can enhance the stem cell properties of tumor cells, which are thought to be resistant to radiotherapy. This study aims to elucidate the potential mechanism of NRP1 in radiation resistance. We transfected NRP1 siRNA and plasmid in breast cancer cells to detect the expression of cancer stem cell markers by western blot and qRT-PCR. The effect of NRP1 on radiotherapy resistance was assesses by immunofluorescence and flow cytometry. In vivo, we established xenograft tumor model treating with shRNA-NRP1 to assess radiotherapy sensitivity. We found that NRP1 could enhance the stem cell properties and confer radioresistance of breast cancer cells. Mechanistically, we proved that NRP1 reduced IR-induced apoptosis by downregulation of Bcl-2 via methyltransferase WTAP in m6A-depentent way. It is suggested that these molecules may be the therapeutic targets for improving the efficacy of radiotherapy for breast cancer.

Alternative splicing of HSPA12A pre-RNA by SRSF11 contributes to metastasis potential of colorectal cancer.

BACKGROUND: Dysregulation of alternative splicing (AS) induced by serine/arginine-rich proteins has recently been linked to cancer metastasis. Nonetheless, as a member of the serine/arginine-rich protein family, the involvement of SRSF11 in colorectal cancer (CRC) is unknown. METHODS: The TCGA dataset and clinical samples were used to assess SRSF11 expression levels in CRC. For SRSF11, functional experiments were conducted both in vitro and in vivo. RNA-seq technology was used to analyze and screen SRSF11-triggered AS events, which were then confirmed by in vivo UV crosslinking and immunoprecipitation (CLIP) and mini-gene reporter assays. Jalview software was used to determine the preferential binding motif with relation to exon skipping (ES) events. Furthermore, coimmunoprecipitation (Co-IP) and Phospho-tag SDS-PAGE experiments were used to investigate PAK5-mediated phosphorylation regulation on SRSF11, and in vitro kinase experiments validated the interaction. RESULTS: In CRC, SRSF11 was discovered to be overexpressed and associated with a poor prognosis. And SRSF11 played a pro-metastatic role in vitro and in vivo. By screening SRSF11-regulated AS events, we identified the binding motif of SRSF11-triggered splicing-switching of HSPA12A AS, which specifically regulated HSPA12A AS by directly binding to a motif in exon 2. Mechanistically, the HSPA12A transcript with exon 2 retention increased N-cadherin expression by promoting RNA stability. Furthermore, the oncogenic kinase PAK5 phosphorylated SRSF11 at serine 287, protecting it from ubiquitination degradation. CONCLUSIONS: SRSF11 exerts pro-metastatic effects in CRC by inhibiting the AS of HSPA12A pre-RNA. Our findings point to SRSF11-regulated HSPA12A splicing as a novel relationship between SRSF11-regulated splicing and CRC metastasis and suggest a PAK5/SRSF11/HSPA12A axis as a potential therapeutic target and prognostic biomarker in CRC.

METTL3 potentiates progression of cervical cancer by suppressing ER stress via regulating m6A modification of TXNDC5 mRNA.

N6-methyladenosine (m6A) is the most abundant chemical modification on mRNA and plays significant roles in many bioprocesses. However, the functions of m6A on cervical cancer (CC) tumorigenesis remain unclear. Here we found methyltransferase-like 3 (METTL3), a core member of the m6A methyltransferase family, was greatly upregulated as an independent prognostic factor in CC. Mechanistically, the transcription factor ETS1 recruited P300 and WDR5 which separately mediated H3K27ac and H3K4me3 histone modification in the promoter of METTL3 and induced METTL3 transcription activation. Furthermore, we identified TXNDC5 as a target of METTL3-mediated m6A modification through MeRIP-seq, and revealed that METTL3-mediated TXNDC5 expression relied on the m6A reader-dependent manner. Functionally, we verified that METTL3 promoted proliferation and metastasis of CC cells by regulating of TXNDC5 expression through in vitro and in vivo experiments. In addition, our study verified the effect of METTL3/TXNDC5 axis on ER stress. Taken together, METTL3 facilitates the malignant progression of CC, suggesting that METTL3 might be a potential prognostic biomarker and therapeutic target for CC.

The Role of Alternative Splicing in Cancer: Regulatory Mechanism, Therapeutic Strategy, and Bioinformatics Application.

[Formula: see text] Alternative splicing (AS) can generate distinct transcripts and subsequent isoforms that play differential functions from the same pre-mRNA. Recently, increasing numbers of studies have emerged, unmasking the association between AS and cancer. In this review, we arranged AS events that are closely related to cancer progression and presented promising treatments based on AS for cancer therapy. Obtaining proliferative capacity, acquiring invasive properties, gaining angiogenic features, shifting metabolic ability, and getting immune escape inclination are all splicing events involved in biological processes. Spliceosome-targeted and antisense oligonucleotide technologies are two novel strategies that are hopeful in tumor therapy. In addition, bioinformatics applications based on AS were summarized for better prediction and elucidation of regulatory routines mingled in. Together, we aimed to provide a better understanding of complicated AS events associated with cancer biology and reveal AS a promising target of cancer treatment in the future.

SHMT2 promotes the tumorigenesis of renal cell carcinoma by regulating the m6A modification of PPAT.

OBJECTIVE: Serine hydroxymethyltransferase 2 (SHMT2) is the first rate-limiting enzyme for serine/glycine biosynthesis and one carbon metabolism. Here, we explore the underlying mechanism of how SHMT2 functions in renal cell carcinoma (RCC) initiation. METHODS: In this study, SHMT2 expression was assessed in RCC tissues. In vitro experiments were performed to investigate the functional role of SHMT2. The detailed mechanisms of SHMT2-mediated PPAT were addressed. RESULTS: Increased SHMT2 facilitated RCC cell proliferation by inducing the G1/S phase transition. And SHMT2 promoted the expression of PPAT. Mechanism dissection revealed that SHMT2 enhanced the m6A modification through the endogenous methyl donor SAM mediated by SHMT2 via serine/glycine one carbon metabolic networks. SHMT2-catalyzed serine/glycine conversion regulated PPAT expression in an m6A-IGF2BP2-dependent manner. SHMT2 promoted RCC cell proliferation by upregulating PPAT expression. CONCLUSIONS: SHMT2 promotes RCC tumorigenesis by increasing PPAT expression. Thus, SHMT2 may be a novel potential therapeutic target for RCC.

MicroRNA-138-1-3p sensitizes sorafenib to hepatocellular carcinoma by targeting PAK5 mediated ß-catenin/ABCB1 signaling pathway.

BACKGROUND: Sorafenib is a kinase inhibitor that is used as a first-line therapy in advanced hepatocellular carcinoma (HCC) patients. However, the existence of sorafenib resistance has limited its therapeutic effect. Through RNA sequencing, we demonstrated that miR-138-1-3p was downregulated in sorafenib resistant HCC cell lines. This study aimed to investigate the role of miR-138-1-3p in sorafenib resistance of HCC. METHODS: In this study, quantitative real-time PCR (qPCR) and Western Blot were utilized to detect the levels of PAK5 in sorafenib-resistant HCC cells and parental cells. The biological functions of miR-138-1-3p and PAK5 in sorafenib-resistant cells and their parental cells were explored by cell viability assays and flow cytometric analyses. The mechanisms for the involvement of PAK5 were examined via co-immunoprecipitation (co-IP), immunofluorescence, dual luciferase reporter assay and chromatin immunoprecipitation (ChIP). The effects of miR-138-1-3p and PAK5 on HCC sorafenib resistant characteristics were investigated by a xenotransplantation model. RESULTS: We detected significant down-regulation of miR-138-1-3p and up-regulation of PAK5 in sorafenib-resistance HCC cell lines. Mechanistic studies revealed that miR-138-1-3p reduced the protein expression of PAK5 by directly targeting the 3'-UTR of PAK5 mRNA. In addition, we verified that PAK5 enhanced the phosphorylation and nuclear translocation of ß-catenin that increased the transcriptional activity of a multidrug resistance protein ABCB1. CONCLUSIONS: PAK5 contributed to the sorafenib resistant characteristics of HCC via ß-catenin/ABCB1 signaling pathway. Our findings identified the correlation between miR-138-1-3p and PAK5 and the molecular mechanisms of PAK5-mediated sorafenib resistance in HCC, which provided a potential therapeutic target in advanced HCC patients.

Serine hydroxymethyltransferase 2: a novel target for human cancer therapy.

Serine and glycine are the primary sources of one-carbon units that are vital for cell proliferation. Their abnormal metabolism is known to be associated with cancer progression. As the key enzyme of serine metabolism, Serine Hydroxymethyltransferase 2 (SHMT2) has been a research hotspot in recent years. SHMT2 is a PLP-dependent tetrameric enzyme that catalyzes the reversible transition from serine to glycine, thus promoting the production of one-carbon units that are indispensable for cell growth and regulation of the redox and epigenetic states of cells. Under a hypoxic environment, SHMT2 can be upregulated and could promote the generation of nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione for maintaining the redox balance. Accumulating evidence confirmed that SHMT2 facilitates cell proliferation and tumor growth and is tightly associated with poor prognosis. In this review, we present insights into the function and research development of SHMT2 and summarize the possible molecular mechanisms of SHMT2 in promoting tumor growth, in the hope that it could provide clues to more effective clinical treatment of cancer.

The biological function of IGF2BPs and their role in tumorigenesis.

The insulin-like growth factor-2 mRNA-binding proteins (IGF2BPs) pertain to a highly conservative RNA-binding family that works as a post-transcriptional fine-tuner for target transcripts. Emerging evidence suggests that IGF2BPs regulate RNA processing and metabolism, including stability, translation, and localization, and are involved in various cellular functions and pathophysiologies. In this review, we summarize the roles and molecular mechanisms of IGF2BPs in cancer development and progression. We mainly discuss the functional relevance of IGF2BPs in embryo development, neurogenesis, metabolism, RNA processing, and tumorigenesis. Understanding IGF2BPs role in tumor progression will provide new insight into cancer pathophysiology.

System Xc-: a key regulatory target of ferroptosis in cancer.

Ferroptosis is a type of oxidative stress-dependent regulated necrosis characterized by excessive lipid peroxide accumulation. This novel cell death modality has been implicated in preventing cancer progression. Cancer cells tend to modulate their redox state to prevent excessive peroxidation, eventually facilitating tumor growth. System Xc- (a cystine/glutamate antiporter system) is a promising target in cancer cells for ferroptosis induction. The overexpression of system Xc-, especially its core subunit xCT, has been reported in several tumors, and these high expression levels were closely related to cancer cell proliferation, invasion, metastasis and the tumor microenvironment. xCT might serve as a novel biomarker, and its upregulation almost always indicates drug tolerance and poor survival. Therefore, system Xc- inhibition may enhance chemotherapy sensitivity and optimize patient prognosis. Here, we elaborate on the mediation of ferroptosis by suppressing system Xc- and the relevant underlying molecular mechanism in cancer cells. The spotlight on this approach to cancer treatment is creating a new horizon and pointing to future opportunities.

N6 -methyladenosine (m6A) RNA modification in human cancer.

N6 -methyladenosine (m6 A) RNA modification, first discovered in 1974, is the most prevalent, abundant and penetrating messenger RNA (mRNA) modification in eukaryotes. This governs the fate of modified transcripts, regulates RNA metabolism and biological processes, and participates in pathogenesis of numerous human diseases, especially in cancer through the reciprocal regulation of m6 A methyltransferases ("writers") and demethylases ("erasers") and the binding proteins decoding m6 A methylation ("readers"). Accumulating evidence indicates a complicated regulation network of m6 A modification involving multiple m6 A-associated regulatory proteins whose biological functions have been further analysed. This review aimed to summarize the current knowledge on the potential significance and molecular mechanisms of m6 A RNA modification in the initiation and progression of cancer.