RITA1 drives the growth of bladder cancer cells by recruiting TRIM25 to facilitate the proteasomal degradation of RBPJ.

Bladder cancer (BC) is one of the most prevalent malignancies worldwide, but it lacks effective targeted therapy due to its elusive molecular mechanism. Therefore, it is important to further investigate the molecular mechanisms that mediate BC progression. By performing a tumor tissue-based gene microarray and shRNA library screening, we found that recombination signal binding protein for immunoglobulin kappa J region (RBPJ) interacting and tubulin associated 1 (RITA1) is crucial for the growth of BC cells. Moreover, RITA1 is aberrantly highly expressed in BC tissues and is also correlated with poor prognosis in patients with BC. Mechanistically, we determined that RITA1 recruits tripartite motif containing 25 (TRIM25) to ubiquitinate RBPJ to accelerate its degradation via proteasome, which leads to the transcriptional inhibition of Notch1 downstream targets. Our results suggest that aberrant high expression of RITA1 drives the growth of BC cells via the RITA1/TRIM25/RBPJ axis and RITA1 may serve as a promising therapeutic target for BC.

Calcium-sensing stromal interaction molecule 2 upregulates nuclear factor of activated T cells 1 and transforming growth factor-ß signaling to promote breast cancer metastasis.

BACKGROUND: Stromal interaction molecule (STIM) 2 is a key calcium-sensing molecule that regulates the stabilization of calcium ions (Ca2+) and therefore regulates downstream Ca2+-associated signaling and cellular events. We hypothesized that STIM2 regulates epithelial-mesenchymal transition (EMT) to promote breast cancer metastasis. METHODS: We determined the effects of gain, loss, and rescue of STIM2 on cellular motility, levels of EMT-related proteins, and secretion of transforming growth factor-ß (TGF-ß). We also conducted bioinformatics analyses and in vivo assessments of breast cancer growth and metastasis using xenograft models. RESULTS: We found a significant association between STIM2 overexpression and metastatic breast cancer. STIM2 overexpression activated the nuclear factor of activated T cells 1 (NFAT1) and TGF-ß signaling. Knockdown of STIM2 inhibited the motility of breast cancer cells by inhibiting EMT via specific suppression of NFAT1 and inhibited mammary tumor metastasis in mice. In contrast, STIM2 overexpression promoted metastasis. These findings were validated in human tissue arrays of 340 breast cancer samples for STIM2. CONCLUSION: Taken together, our results demonstrated that STIM2 specifically regulates NFAT1, which in turn regulates the expression and secretion of TGF-ß1 to promote EMT in vitro and in vivo, leading to metastasis of breast cancer.

Exosomes derived from breast cancer lung metastasis subpopulations promote tumor self-seeding.

Lung metastasis is a primary obstacle in the clinical treatment of metastatic breast cancer. Most patients with lung metastasis eventually die of recurrence. Recurrence may be related to self-seeding, which occurs when circulating tumor cells re-seed into the tumors they originated from (metastasis or carcinoma in situ). Tumor-derived exosomes have been intensively revealed to promote the progression of various cancers. However, whether tumor-derived exosomes play roles in tumor self-seeding has not yet been identified. By establishing a self-seeding nude mouse model, we found that exosomes derived from MDA231-LM2 cells (subpopulations of breast cancer lung metastasis) potentiate the growth of MDA-MB-231 xenografts. More importantly, laser confocal microscopy and flow cytometry results identified that MDA231-LM2-secreted exosomes promote the seeding of MDA231-LM2 cells into MDA-MB-231 xenografts. These findings suggest MDA231-LM2-secreted exosomes as a promising target to treat breast cancer lung metastasis.

STIM1 and STIM2 differently regulate endogenous Ca2+ entry and promote TGF-ß-induced EMT in breast cancer cells.

The Ca2+ sensor proteins STIM1 and STIM2 are crucial elements of store-operated calcium entry (SOCE) in breast cancer cells. Increased SOCE activity may contribute to epithelial-mesenchymal transitions (EMT) and increase cell migration and invasion. However, the roles of STIM1 and STIM2 in TGF-ß-induced EMT are still unclear. In this study, we demonstrate roles of STIMs in TGF-ß-induced EMT in breast cancer cells. In particular, STIM1 and STIM2 expression affected TGF-ß-induced EMT by mediating SOCE in MDA-MB-231 and MCF-7 breast cancer cells. The specific SOCE inhibitor YM58483 blocked TGF-ß-induced EMT, and differing effects of STIM1 and STIM2 on TGF-ß-induced EMT correlated with differing roles in SOCE. Finally, we showed that STIM2 is associated with non-store-operated calcium entry (non-SOCE) during TGF-ß-induced EMT, whereas STIM1 is not. What's more, non-SOCE have a large possibility to be ROCE. In conclusion, STIM1 and STIM2 proteins play important roles in TGF-ß-induced EMT and these effects are related to both SOCE and non-SOCE.

G3BP1 and SLU7 Jointly Promote Immune Evasion by Downregulating MHC-I via PI3K/Akt Activation in Bladder Cancer.

Immune checkpoint inhibitors (ICIs) show promise as second-line treatment for advanced bladder cancer (BLCA); however, their responsiveness is limited by the immune evasion mechanisms in tumor cells. This study conduct a Cox regression analysis to screen mRNA-binding proteins and reveals an association between Ras GTPase-activating protein-binding protein 1 (G3BP1) and diminished effectiveness of ICI therapy in patients with advanced BLCA. Subsequent investigation demonstrates that G3BP1 enhances immune evasion in BLCA cells by downregulating major histocompatibility complex class I (MHC-I) through phosphoinositide 3-kinase (PI3K)/Akt signaling activation. Mechanistically, G3BP1 interacts with splicing factor synergistic lethal with U5 snRNA 7 (SLU7) to form a complex with poly(A)-binding protein cytoplasmic 1 and eukaryotic translation initiation factor 4 gamma 1. This complex stabilizes the closed-loop structure of the mRNAs of class IA PI3Ks and consequently facilitates their translation and stabilization, thereby activating PI3K/Akt signaling to downregulate MHC-I. Consistently, targeting G3BP1 with epigallocatechin gallate (EGCG) impedes immune evasion and sensitizes BLCA cells to anti-programmed cell death (PD)-1 antibodies in mice. Thus, G3BP1 and SLU7 collaboratively contribute to immune evasion in BLCA, indicating that EGCG is a precision therapeutic agent to enhance the effectiveness of anti-PD-1 therapy.

Targeting the KAT8/YEATS4 Axis Represses Tumor Growth and Increases Cisplatin Sensitivity in Bladder Cancer.

Bladder cancer (BC) is one of the most common tumors characterized by a high rate of relapse and a lack of targeted therapy. Here, YEATS domain-containing protein 4 (YEATS4) is an essential gene for BC cell viability using CRISPR-Cas9 library screening is reported, and that HUWE1 is an E3 ligase responsible for YEATS4 ubiquitination and proteasomal degradation by the Protein Stability Regulators Screening Assay. KAT8-mediated acetylation of YEATS4 impaired its interaction with HUWE1 and consequently prevented its ubiquitination and degradation. The protein levels of YEATS4 and KAT8 are positively correlated and high levels of these two proteins are associated with poor overall survival in BC patients. Importantly, suppression of YEATS4 acetylation with the KAT8 inhibitor MG149 decreased YEATS4 acetylation, reduced cell viability, and sensitized BC cells to cisplatin treatment. The findings reveal a critical role of the KAT8/YEATS4 axis in both tumor growth and cisplatin sensitivity in BC cells, potentially generating a novel therapeutic strategy for BC patients.

NFYC-37 promotes tumor growth by activating the mevalonate pathway in bladder cancer.

Dysregulation of transcription is a hallmark of cancer, including bladder cancer (BLCA). CRISPR-Cas9 screening using a lentivirus library with single guide RNAs (sgRNAs) targeting human transcription factors and chromatin modifiers is used to reveal genes critical for the proliferation and survival of BLCA cells. As a result, the nuclear transcription factor Y subunit gamma (NFYC)-37, but not NFYC-50, is observed to promote cell proliferation and tumor growth in BLCA. Mechanistically, NFYC-37 interacts with CBP and SREBP2 to activate mevalonate pathway transcription, promoting cholesterol biosynthesis. However, NFYC-50 recruits more of the arginine methyltransferase CARM1 than NFYC-37 to methylate CBP, which prevents the CBP-SREBP2 interaction and subsequently inhibits the mevalonate pathway. Importantly, statins targeting the mevalonate pathway can suppress NFYC-37-induced cell proliferation and tumor growth, indicating the need for conducting a clinical trial with statins for treating patients with BLCA and high NFYC-37 levels, as most patients with BLCA have high NFYC-37 levels.

Downregulation of HINFP induces senescence-associated secretory phenotype to promote metastasis in a non-cell-autonomous manner in bladder cancer.

Transcription dysregulation is a salient characteristic of bladder cancer (BC), but no appropriate therapeutic target for it has been established. Here, we found that heterogeneous downregulation of histone H4 transcription factor (HINFP) was associated with senescence in BC tissues and that lower HINFP expression could predict an unfavorable outcome in BC patients. Knockout of HINFP transcriptionally inhibited H1F0 and H1FX to trigger DNA damage, consequently inducing cell senescence to repress the proliferation and growth of BC cells. However, the senescence-associated secretory phenotype, characterized by increases in MMP1/3, enhances the invasion and metastasis of non-senescent BC cells. Histone deacetylase inhibitors (HDACis) could efficiently eliminate the senescent cells induced by HINFP knockout to suppress the invasion and metastasis of BC cells. Our study suggests that HDACis, widely used in multiple cancer types in a clinical context, may also benefit BC patients with metastases induced by cell senescence.

FXR1 can bind with the CFIm25/CFIm68 complex and promote the progression of urothelial carcinoma of the bladder by stabilizing TRAF1 mRNA.

RNA-binding proteins (RBPs) are key regulators of gene expression. RBP dysregulation is reported to play essential roles in tumorigenesis. However, the role of RBPs in urothelial carcinoma of the bladder (UCB) is only starting to be unveiled. Here, we comprehensively assessed the mRNA expression landscape of 104 RBPs from two independent UCB cohorts, Sun Yat-sen University Cancer Center (SYSUCC) and The Cancer Genome Atlas (TCGA). Fragile X-related gene 1 (FXR1) was identified as a novel cancer driver gene in UCB. FXR1 overexpression was found to be related to the poor survival rate in the SYSUCC and TCGA cohorts. Functionally, FXR1 promotes UCB proliferation and tumorigenesis. Mechanistically, FXR1 serves as a platform to recruit CFIm25 and CFIm68, forming a novel 3' processing machinery that functions in sequence-specific poly(A) site recognition. FXR1 affects the 3' processing of Tumor necrosis factor receptor-associated factor 1 (TRAF1) mRNA, which leads to nuclear stabilization. The novel regulatory relationship between FXR1 and TRAF1 can enhance cell proliferation and suppress apoptosis. Our data collectively highlight the novel regulatory role of FXR1 in TRAF1 3' processing as an important determinant of UCB oncogenesis. Our study provides new insight into RBP function and provides a potential therapeutic target for UCB.

Knockdown of ZBTB11 impedes R-loop elimination and increases the sensitivity to cisplatin by inhibiting DDX1 transcription in bladder cancer.

INTRODUCTION: Bladder cancer (BC) is one of the most common malignant cancers, with poor prognosis and high incidence. Cisplatin is the standard chemotherapy for muscle invasive bladder cancer; however, chemotherapy resistance remains a major challenge. Moreover, oncogenic signalling and the specific mechanisms underlying cisplatin resistance in BC remain largely unclear METHODS: In this study, RT-PCR, Western blot, immunofluorescence, and immunohistochemistry were used to measure gene and protein expression. Colony formation assay and flow cytometry were performed to evaluate the proliferation of BC cells. Gene set enrichment analysis was performed to identify the function in which ZBTB11 was involved. Luciferase and chromatin immunoprecipitation experiments were performed to determine the transcriptional regulation mechanism of ZBTB11. The effects of ZBTB11 on the malignant phenotypes of BC cells were examined in vitro and in vivo RESULTS: The results showed that ZBTB11 was remarkably upregulated in BC tissues, which was associated with poor prognosis in patients with BC. Furthermore, we found that knockdown of ZBTB11 remarkably inhibited the proliferation and tumorigenesis of BC cells by inducing apoptosis. Mechanistically, the knockdown of ZBTB11 transcriptionally inhibited DDX1 to suppress R-loop clearance, resulting in DNA damage in BC cells. Importantly, the ZBTB11/DDX1 axis is required for the chemotherapy resistance of BC cells to cisplatin CONCLUSION: Our findings not only reveal an underlying mechanism by which the ZBTB11/DDX1 axis promotes the tumorigenesis of BC but also provide a potential target for a combination strategy of cisplatin-based chemotherapy for BC.

MCU-dependent negative sorting of miR-4488 to extracellular vesicles enhances angiogenesis and promotes breast cancer metastatic colonization.

Based on Stephen Paget's well-established theory, both cell-autonomous and non-cell-autonomous mechanisms are crucial for metastasis. Although the mitochondrial calcium uniporter (MCU) has been suggested to be involved in breast cancer (BC) progression via cell-autonomous mechanisms, whether it assists the metastasis of BC cells through non-cell-autonomous mechanisms remains unclear. This study aimed to demonstrate that the MCU regulates BC metastatic colonization via non-cell-autonomous mechanisms. The results suggested that extracellular vesicles (EVs) derived from MCU-downregulated MDA-MB-231 cells suppressed angiogenesis in the metastatic niche in a nude mouse model, thereby hindering the colonization of BC cells. Mechanistically, we revealed that the MCU negatively correlated with miR-4488 in EVs derived from BC cells. Significantly, miR-4488 was determined to suppress angiogenesis of vascular endothelial cells by directly targeting angiogenic CX3CL1. Furthermore, we identified miR-4488 as being significantly downregulated in serum EVs from patients with triple-negative BC. Hence, this study suggests that MCU-dependent negative sorting of miR-4488 to EVs enhances angiogenesis in the metastatic niche and, thus, favors the metastatic colonization of BC cells.

Knockdown of FAM64A suppresses proliferation and migration of breast cancer cells.

BACKGROUND: FAM64A is a mitotic regulator promoting cell metaphase-anaphase transition, and it is frequently reported to be highly expressed in cancer cells. However, the role of FAM64A in human breast cancer (BrC) is poorly studied. METHODS: The expression of FAM64A mRNA in BrC samples was determined by RT-qPCR assay and TCGA database mining. Kaplan-Meier plotter was used to analyze whether FAM64A expression impacted prognosis. Then, the expression of FAM64A was silenced using RNA interference. Cell-counting assay, colony formation assay and flow cytometry assay were conducted to detect proliferation; transwell migration assay, EMT-related proteins expression (E-cadherin, N-cadherin and vimentin), and EMT-related transcription factors mRNA expression (Snail, Twist, Slug) were conducted to evaluate the migration ability. RESULTS: FAM64A was highly expressed in human BrC samples, which was negatively associated with poor survival time. Analysis of FAM64A expression in BrC cell lines demonstrated that the expression of FAM64A was significantly correlated with the proliferation rate and migration ability of BrC cells. Indeed, knockdown of FAM64A suppressed the proliferation of MDA-MB-231 and MCF-7 cells. Importantly, we also found that silencing of FAM64A inhibited the migration of BrC cells via impeding epithelial-mesenchymal transition. CONCLUSIONS: Our findings suggest that FAM64A plays an important role in the proliferation and migration of BrC cells, which might serve as a potential target for BrC treatment.

Phosphorylation of dynamin-related protein 1 at Ser616 regulates mitochondrial fission and is involved in mitochondrial calcium uniporter-mediated neutrophil polarization and chemotaxis.

During an inflammatory response, polarization of neutrophils is necessary for effective chemotaxis and bacterial endocytosis. Ca2+ uptake into mitochondria through the mitochondrial calcium uniporter (MCU) is crucial for cell metabolism, signaling and survival; however, the physiological role of MCU in human neutrophils remains unclear. Here we show that MCU is vital for the polarization and chemotaxis of neutrophils. Activation of MCU by spermine promotes neutrophil polarization and chemotaxis, whereas inhibition of MCU by Ru360 blunts both processes. We also provide evidence that this role of the MCU in neutrophils may result from modulation of mitochondrial fission by increased levels of pDrp1 S616 via accumulation of Ca2+ into the mitochondrial matrix. Thus, our study identifies the dependence of neutrophil polarization and chemotaxis on the MCU and highlights the importance of regulating mitochondrial fission during the anti-inflammatory cascade in human neutrophils.