HnRNPA2B1 ISGylation Regulates m6A-Tagged mRNA Selective Export via ALYREF/NXF1 Complex to Foster Breast Cancer Development.

Regulating nuclear export precisely is essential for maintaining mRNA homeostasis and impacts tumor progression. However, the mechanisms governing nuclear mRNA export remain poorly elucidated. Herein, it is revealed that the enhanced hypoxic long no-ncoding RNA (lncRNA prostate cancer associated transcript 6 (PCAT6) in breast cancer (BC) promotes the nuclear export of m6A-modified mRNAs, bolstering breast cancer stem cells (BCSCs) stemness and doxorubicin resistance. Clinically, hypoxic PCAT6 correlates with malignant BC features and poor prognosis. Mechanically, PCAT6 functions as a scaffold between interferon-stimulated gene 15 (ISG15) and heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1), leading to ISGylation of hnRNPA2B1, thus protecting hnRNPA2B1 from ubiquitination-mediated proteasomal degradation. Interestingly, as an m6A reader, hnRNPA2B1 selectively mediates m6A-tagged mRNAs nuclear export via the Aly/REF export factor (ALYREF)/ nuclear RNA export factor 1 (NXF1) complex, which promotes stemness-related genes expression. HnRNPA2B1 knockdown or mRNA export inhibition can result in the retention of nuclear m6A-tagged mRNA associated with stemness maintenance, which suppresses BCSCs self-renewal and effectively improves the efficacy of doxorubicin therapy. These findings demonstrate the pivotal role of m6A-modified mRNA nuclear export in BC progression, highlighting that the inhibition of m6A-tagged mRNA and its nuclear export is a potential therapeutic strategy for the amelioration of cancer chemotherapy.

ACSL4-mediated membrane phospholipid remodeling induces integrin ß1 activation to facilitate triple-negative breast cancer metastasis.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer and has a poor prognosis and a high propensity to metastasize. Lipid metabolism has emerged as a critical regulator of tumor progression and metastasis in other cancer types. Characterization of the lipid metabolic features of TNBC could provide important insights into the drivers of TNBC metastasis. Here, we showed that metastatic TNBC tumors harbor more unsaturated phospholipids, especially long-chain polyunsaturated fatty acids, at the sn-2 position of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) compared to primary tumors. Metastatic TNBC tumors upregulated ACSL4, a long-chain polyunsaturated acyl-CoA synthetase that drives the preferential incorporation of polyunsaturated fatty acids into phospholipids, resulting in the alteration of membrane phospholipid composition and properties. Moreover, ACSL4-mediated phospholipid remodeling of the cell membrane induced lipid-raft localization and activation of integrin ß1 in a CD47-dependent manner, which led to downstream focal adhesion kinase (FAK) phosphorylation that promoted metastasis. Importantly, pharmacological inhibition of ACSL4 suppressed tumor growth and metastasis and increased chemosensitivity in TNBC models in vivo. These findings indicate that ACSL4-mediated phospholipid remodeling enables TNBC metastasis and can be inhibited as a potential strategy to improve the efficacy of chemotherapy in TNBC.

Oxidized ATM governs stemness of breast cancer stem cell through regulating ubiquitylation and acetylation switch.

Cancer stem cells (CSCs), as parts of tumor initiation cells, play a crucial role to tumorigenesis, development and recurrence. However, the complicated mechanisms of CSCs to adapt to tumor microenvironment and its stemness maintenance remains unclear. Here, we show that oxidized ATM, a hypoxia-activated cytoplasm ATM, acts a novel function to maintain CSC stemness in triple-negative breast cancer cells (BCSCs) via regulating histone H4 acetylation. Mechanistically, oxidized ATM phosphorylates TRIM21 (a E3 ubiquitin ligase) serine 80 and serine 469. Serine 80 phosphorylation of TRIM21 is essential for the ubiquitination activity of TRIM21. TRIM21 binds with SIRT1 (one of deacetylase), resulting in ubiquitylation-mediated degradation of SIRT1. The reduced SIRT1 leads to increase of histone H4 acetylation, thus facilitating CSC-related gene expression. Clinical data verify that high level of ATM in breast tumors is positively correlated with malignant grade, and is closely related with low SIRT1, high p-TRIM21, and high CD44 expression. In conclusion, our study provides a novel mechanism by which oxidized ATM governing BCSCs stemness and reveals an important link among oxidized ATM, histone acetylation, and BCSCs maintenance.

RGCC-mediated PLK1 activity drives breast cancer lung metastasis by phosphorylating AMPKα2 to activate oxidative phosphorylation and fatty acid oxidation.

BACKGROUND: More than 90% of the mortality of triple-negative breast cancer (TNBC) patients is attributed to cancer metastasis with organotropism. The lung is a frequent site of TNBC metastasis. However, the precise molecular mechanism for lung-specific metastasis of TNBC is not well understood. METHODS: RNA sequencing was performed to identify patterns of gene expression associated with lung metastatic behavior using 4T1-LM3, MBA-MB-231-LM3, and their parental cells (4T1-P, MBA-MB-231-P). Expressions of RGCC, called regulator of cell cycle or response gene to complement 32 protein, were detected in TNBC cells and tissues by qRT-PCR, western blotting, and immunohistochemistry. Kinase activity assay was performed to evaluate PLK1 kinase activity. The amount of phosphorylated AMP-activated protein kinase α2 (AMPKα2) was detected by immunoblotting. RGCC-mediated metabolism was determined by UHPLC system. Oxidative phosphorylation was evaluated by JC-1 staining and oxygen consumption rate (OCR) assay. Fatty acid oxidation assay was conducted to measure the status of RGCC-mediated fatty acid oxidation. NADPH and ROS levels were detected by well-established assays. The chemical sensitivity of cells was evaluated by CCK8 assay. RESULTS: RGCC is aberrantly upregulated in pulmonary metastatic cells. High level of RGCC is significantly related with lung metastasis in comparison with other organ metastases. RGCC can effectively promote kinase activity of PLK1, and the activated PLK1 phosphorylates AMPKα2 to facilitate TNBC lung metastasis. Mechanistically, the RGCC/PLK1/AMPKα2 signal axis increases oxidative phosphorylation of mitochondria to generate more energy, and promotes fatty acid oxidation to produce abundant NADPH. These metabolic changes contribute to sustaining redox homeostasis and preventing excessive accumulation of potentially detrimental ROS in metastatic tumor cells, thereby supporting TNBC cell survival and colonization during metastases. Importantly, targeting RGCC in combination with paclitaxel/carboplatin effectively suppresses pulmonary TNBC lung metastasis in a mouse model. CONCLUSIONS: RGCC overexpression is significantly associated with lung-specific metastasis of TNBC. RGCC activates AMPKα2 and downstream signaling through RGCC-driven PLK1 activity to facilitate TNBC lung metastasis. The study provides implications for RGCC-driven OXPHOS and fatty acid oxidation as important therapeutic targets for TNBC treatment.

Hypoxia-induced circSTT3A enhances serine synthesis and promotes H3K4me3 modification to facilitate breast cancer stem cell formation.

Hypoxia is a key feature of tumor microenvironment that contributes to the development of breast cancer stem cells (BCSCs) with strong self-renewal properties. However, the specific mechanism underlying hypoxia in BCSC induction is not completely understood. Herein, we provide evidence that a novel hypoxia-specific circSTT3A is significantly upregulated in clinical breast cancer (BC) tissues, and is closely related to the clinical stage and poor prognosis of patients with BC. The study revealed that hypoxia-inducible factor 1 alpha (HIF1α)-regulated circSTT3A has a remarkable effect on mammosphere formation in breast cancer cells. Mechanistically, circSTT3A directly interacts with nucleotide-binding domain of heat shock protein 70 (HSP70), thereby facilitating the recruitment of phosphoglycerate kinase 1 (PGK1) via its substrate-binding domain, which reduces the ubiquitination and increases the stability of PGK1. The enhanced levels of PGK1 catalyze 1,3-diphosphoglycerate (1,3-BPG) into 3-phosphoglycerate (3-PG) leading to 3-PG accumulation and increased serine synthesis, S-adenosylmethionine (SAM) accumulation, and trimethylation of histone H3 lysine 4 (H3K4me3). The activation of the H3K4me3 contributes to BCSCs by increasing the transcriptional level of stemness-related factors. Especially, our work reveals that either loss of circSTT3A or PGK1 substantially suppresses tumor initiation and tumor growth, which dramatically increases the sensitivity of tumors to doxorubicin (DOX) in mice. Injection of PGK1-silenced spheroids with 3-PG can significantly reverse tumor initiation and growth in mice, thereby increasing tumor resistance to DOX. In conclusion, our study sheds light on the functional role of hypoxia in the maintenance of BCSCs via circSTT3A/HSP70/PGK1-mediated serine synthesis, which provides new insights into metabolic reprogramming, tumor initiation and growth. Our findings suggest that targeting circSTT3A alone or in combination with chemotherapy has potential clinical value for BC management.

TCF12 regulates exosome release from epirubicin-treated CAFs to promote ER+ breast cancer cell chemoresistance.

Cancer-associated fibroblasts (CAFs) are the predominant stromal cells in the microenvironment and play important roles in tumor progression, including chemoresistance. However, the response of CAFs to chemotherapeutics and their effects on chemotherapeutic outcomes are largely unknown. In this study, we showed that epirubicin (EPI) treatment triggered ROS which initiated autophagy in CAFs, TCF12 inhibited autophagy flux and further promoted exosome secretion. Inhibition of EPI-induced reactive oxygen species (ROS) production with N-acetyl-L-cysteine (NAC) or suppression of autophagic initiation with short interfering RNA (siRNA) against ATG5 blunted exosome release from CAFs. Furthermore, exosome secreted from EPI-treated CAFs not only prevented ROS accumulation in CAFs but also upregulated the CXCR4 and c-Myc protein levels in recipient ER+ breast cancer cells, thus promoting EPI resistance of tumor cells. Together, the current study provides novel insights into the role of stressed CAFs in promoting tumor chemoresistance and reveal a new function of TCF12 in regulating autophagy impairment and exosome release.

Cancer-associated fibroblasts facilitate premetastatic niche formation through lncRNA SNHG5-mediated angiogenesis and vascular permeability in breast cancer.

Background: Metastasis is the leading cause of death in patients with breast cancer (BC). Primary tumors create a premetastatic niche (PMN) in secondary organs for subsequent metastases. Cancer-associated fibroblasts (CAFs) are a predominant stromal component in the tumor microenvironment and serve as a major contributor to tumor metastasis. However, the function and mechanism of primary CAFs in the premetastatic niche of secondary organs remain unclear in BC. Methods: We investigated the expression profiles of lncRNAs in pairs of CAFs and NFs derived from breast tumor tissues using lncRNA microarray. The expression levels of lncSNHG5, ZNF281, IGF2BP2, CCL2 and CCL5 were assessed by qRT-PCR; the protein levels of related genes (e.g., ZNF281, IGF2BP2, CCL2, and CCL5) were analyzed using western blotting and/or ELISA in primary and immortalized CAFs and clinical samples. Tubule formation and three-dimensional sprouting assays and tissue fluorescence staining were conducted to investigate angiogenesis. In vitro permeability assays, trans-endothelial invasion assays, in vivo permeability assays and tissue fluorescence staining were conducted to examine vascular permeability. The regulatory mechanism of lncSNHG5 was investigated by RNA sequencing, fluorescent in situ hybridization, cellular fractionation assay, mass spectrometry, RNA pull-down, RNA immunoprecipitation, gene-specific m6A assay, chromatin immunoprecipitation, dual luciferase reporter assay and actinomycin D treatment in CAFs and NFs. Results: LncSNHG5 was highly expressed in breast CAFs and played an essential role in premetastatic niche formation by promoting angiogenesis and vascular leakiness through regulation of ZNF281 in CAFs. lncSNHG5 enhanced ZNF281 mRNA stability by binding with the m6A reader IGF2BP2. Enhanced ZNF281 transcriptionally regulated CCL2 and CCL5 expression to activate P38 MAPK signaling in endothelial cells. High CCL2 and CCL5 expression was associated with tumor metastasis and poor prognosis in BC patients. The inhibitors RS102895, marasviroc and cenicriviroc inhibited angiogenesis and vascular permeability in the PMN by blocking the binding of CCL2/CCR2 and CCL5/CCR5. The lncSNHG5-ZNF281-CCL2/CCL5 signaling axis plays an essential role in inducing premetastatic niche formation to promote BC metastasis. Conclusions: Our work demonstrates that lncSNHG5 and its downstream signaling ZNF281-CCL2/CCL5 in CAFs play a crucial role in premetastatic niche formation in breast cancer and may serve as potential targets for the diagnosis and treatment of BC metastasis.

Breast cancer liver metastasis: Pathogenesis and clinical implications.

Breast cancer is the most common malignant disease in female patients worldwide and can spread to almost every place in the human body, most frequently metastasizing to lymph nodes, bones, lungs, liver and brain. The liver is a common metastatic location for solid cancers as a whole, and it is also the third most common metastatic site for breast cancer. Breast cancer liver metastasis (BCLM) is a complex process. Although the hepatic microenvironment and liver sinusoidal structure are crucial factors for the initial arrest of breast cancer and progression within the liver, the biological basis of BCLM remains to be elucidated. Importantly, further understanding of the interaction between breast cancer cells and hepatic microenvironment in the liver metastasis of breast cancer will suggest ways for the development of effective therapy and prevention strategies for BCLM. In this review, we provide an overview of the recent advances in the understanding of the molecular mechanisms of the hepatic microenvironment in BCLM formation and discuss current systemic therapies for treating patients with BCLM as well as potential therapeutic development based on the liver microenvironment-associated signaling proteins governing BCLM.

The Drosha-Independent MicroRNA6778-5p/GSK3ß Axis Mediates the Proliferation of Gastric Cancer Cells.

Background: Gastric cancer (GC) is a primary cause of cancer death around the world. Previous studies have found that Drosha plays a significant role in the development of tumor cells. Soon after, we unexpectedly found that the expression of microRNA6778-5p (miR6778-5p) is unconventionally high in the gastric cancer cells low-expressing Drosha. So, we designed the Drosha interference sequence and recombined it into a lentiviral vector to construct Drosha knockdown lentivirus and transfected the Drosha knockdown lentivirus into gastric cancer cells to establish Drosha knockdown gastric cancer cell lines. We aimed to explore the effect of microRNA6778-5p on the proliferation of gastric cancer cells with Drosha knockdown and its intrinsic mechanism. Methods: We designed the Drosha interference sequence and recombined it into a lentiviral vector to construct Drosha knockdown lentivirus and transfected the Drosha knockdown lentivirus into gastric cancer cells to establish Drosha knockdown gastric cancer cell lines. After transfecting miR6778-5p mimics and inhibitor into gastric cancer cell lines with Drosha knockdown, the expression levels of miR6778-5p mimics in Drosha low-expressing gastric cancer cells increased, while miR6778-5p inhibitor decreased the expression levels of miR6778-5p. The Cell Counting Kit-8 (CCK-8) experiment was used to detect the proliferation ability of gastric cancer cells after overexpression or knockdown of miR6778-5p and bioinformatics predicted the relationship between miR6778-5p and glycogen synthase kinase-3ß (GSK3ß). Results: After infection with the Drosha knockdown lentivirus, Drosha's mRNA and protein levels were significantly downregulated in gastric cancer cells. The expression levels of miR6778-5p mimics in Drosha low-expressing gastric cancer cells increased, while miR6778-5p inhibitor decreased the expression levels of miR6778-5p. Overexpression of miR6778-5p significantly enhanced the proliferation ability of Drosha low-expression gastric cancer cells; on the contrary, knocking down miR6778-5p weakened the proliferation ability of Drosha low-expression gastric cancer cells. Bioinformatics predicted that miR6778-5p targeted glycogen synthase kinase-3ß (GSK3ß) and the mRNA and protein levels of GSK3ß decreased significantly after overexpression of miR6778-5p. Conclusion: miR6778-5p promotes the proliferation of Drosha low-expressing gastric cancer cells by targeting GSK3ß.

Erratum: FOSL2 promotes VEGF-independent angiogenesis by transcriptionnally activating Wnt5a in breast cancer-associated fibroblasts: Erratum.

[This corrects the article DOI: 10.7150/thno.55074.].

A novel lncRNA ROPM-mediated lipid metabolism governs breast cancer stem cell properties.

BACKGROUND: Cancer stem cells (CSCs) are considered as the major cause to tumor initiation, recurrence, metastasis, and drug resistance, driving poor clinical outcomes in patients. Long noncoding RNAs (lncRNAs) have emerged as crucial regulators in cancer development and progression. However, limited lncRNAs involved in CSCs have been reported. METHODS: The novel lncROPM (a regulator of phospholipid metabolism) in breast CSCs (BCSCs) was identified by microarray and validated by qRT-PCR in BCSCs from breast cancer cells and tissues. The clinical significance of lncROPM was evaluated in two breast cancer cohorts and TANRIC database (TCGA-BRCA, RNAseq data). Gain- and loss-of-function assays were performed to examine the role of lncROPM on BCSCs both in vitro and in vivo. The regulatory mechanism of lncROPM was investigated by bioinformatics, RNA FISH, RNA pull-down, luciferase reporter assay, and actinomycin D treatment. PLA2G16-mediated phospholipid metabolism was determined by UHPLC-QTOFMS system. Cells' chemosensitivity was assessed by CCK8 assay. RESULTS: LncROPM is highly expressed in BCSCs. The enhanced lncROPM exists in clinic breast tumors and other solid tumors and positively correlates with malignant grade/stage and poor prognosis in breast cancer patients. Gain- and loss-of-function studies show that lncROPM is required for the maintenance of BCSCs properties both in vitro and in vivo. Mechanistically, lncROPM regulates PLA2G16 expression by directly binding to 3'-UTR of PLA2G16 to increase the mRNA stability. The increased PLA2G16 significantly promotes phospholipid metabolism and the production of free fatty acid, especially arachidonic acid in BCSCs, thereby activating PI3K/AKT, Wnt/ß-catenin, and Hippo/YAP signaling, thus eventually involving in the maintenance of BCSCs stemness. Importantly, lncROPM and PLA2G16 notably contribute to BCSCs chemo-resistance. Administration of BCSCs using clinic therapeutic drugs such as doxorubicin, cisplatin, or tamoxifen combined with Giripladib (an inhibitor of cytoplasmic phospholipase A2) can efficiently eliminate BCSCs and tumorigenesis. CONCLUSIONS: Our study highlights that lncROPM and its target PLA2G16 play crucial roles in sustaining BCSC properties and may serve as a biomarker for BCSCs or other cancer stem cells. Targeting lncROPM-PLA2G16 signaling axis may be a novel therapeutic strategy for patients with breast cancer.

A pseudo-targeted metabolomics study based on serum bile acids profiling for the differential diagnosis of benign and malignant breast lesions.

INTRODUCTION: Breast cancer (BC) has become the most commonly diagnosed cancer worldwide. It is very critical for the differential diagnosis between BC and benign breast diseases (BBD). The characteristics of serum bile acids (BAs) profiling in patients with BBD and BC was elucidated so that potential biomarkers could be find out for the differential diagnosis of BC and BBD. METHODS: A pseudo-targeted approach was used to perform BAs metabolomics analysis in serum of 29 patients with BBD and 47 patients with BC by ultra-high performance liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). Partial least squares-discriminant analysis (PLS-DA) was used to establish a differential diagnostic model for BC, and the receiver operating characteristic (ROC) curve and logistic regression analysis were used to screen out bile acids as biomarkers for the differential diagnosis of BC and BBD. RESULTS: The serum BAs profile in BC group was quite different from that in BBD group. Compared with the BBD group, BC group had higher level of chenodeoxycholic acid (CDCA), while they had lower levels of dihydroxy tauro-conjugated BA (Tdi-1) and sulfated dihydroxy glyco-conjugated BA (Gdi-S-1). The sensitivity and specificity of PLS-DA model for patients classification were 100% and 92.3%, respectively. The combined biomarker, CDCA and Tdi-1, had high efficacy for the differential diagnosis (area under the curve was 0.954, 95% CI: 0.880-1.000) of BC. Besides, the performance was superior to traditional biomarkers in the differential diagnosis of BC with or without comorbidities. CONCLUSION: The profile of serum BAs in women with BC was quite different from that in patients with BBD. Serum BAs profiling analysis could be used as an effective tool for the differential diagnosis of BC and BBD.

Hypoxia-stimulated ATM activation regulates autophagy-associated exosome release from cancer-associated fibroblasts to promote cancer cell invasion.

Cancer-associated fibroblasts (CAFs) as a predominant cell component in the tumour microenvironment (TME) play an essential role in tumour progression. Our earlier studies revealed oxidized ATM activation in breast CAFs, which is independent of DNA double-strand breaks (DSBs). Oxidized ATM has been found to serve as a redox sensor to maintain cellular redox homeostasis. However, whether and how oxidized ATM in breast CAFs regulates breast cancer progression remains poorly understood. In this study, we found that oxidized ATM phosphorylates BNIP3 to induce autophagosome accumulation and exosome release from hypoxic breast CAFs. Inhibition of oxidized ATM kinase by KU60019 (a small-molecule inhibitor of activated ATM) or shRNA-mediated knockdown of endogenous ATM or BNIP3 blocks autophagy and exosome release from hypoxic CAFs. We also show that oxidized ATM phosphorylates ATP6V1G1, a core proton pump in maintaining lysosomal acidification, leading to lysosomal dysfunction and autophagosome fusion with multi-vesicular bodies (MVB) but not lysosomes to facilitate exosome release. Furthermore, autophagy-associated GPR64 is enriched in hypoxic CAFs-derived exosomes, which stimulates the non-canonical NF-κB signalling to upregulate MMP9 and IL-8 in recipient breast cancer cells, enabling cancer cells to acquire enhanced invasive abilities. Collectively, these results provide novel insights into the role of stromal CAFs in promoting tumour progression and reveal a new function of oxidized ATM in regulating autophagy and exosome release.

Long non-coding RNA Lnc-408 promotes invasion and metastasis of breast cancer cell by regulating LIMK1.

Invasion and metastasis are the leading causes of death in patients with breast cancer (BC), and epithelial-mesenchymal transformation (EMT) plays an essential role in this process. Here, we found that Lnc-408, a novel long noncoding RNA (lncRNA), is significantly upregulated in BC cells undergoing EMT and in BC tumor with lymphatic metastases compared with those without lymphatic metastases. Lnc-408 can enhance BC invasion and metastasis by regulating the expression of LIMK1. Mechanistically, Lnc-408 serves as a sponge for miR-654-5p to relieve the suppression of miR-654-5p on its target LIMK1. Knockdown or knockout of Lnc-408 in invasive BC cells clearly decreased LIMK1 levels, and ectopic Lnc-408 in MCF-7 cells increased LIMK1 expression to promote cell invasion. Lnc-408-mediated enhancement of LIMK1 plays a key role in cytoskeletal stability and promotes invadopodium formation in BC cells via p-cofilin/F-actin. In addition, the increased LIMK1 also facilitates the expression of MMP2, ITGB1, and COL1A1 by phosphorylating CREB. In conclusion, our findings reveal that Lnc-408 promotes BC invasion and metastasis via the Lnc-408/miR-654-5p/LIMK1 axis, highlighting a novel promising target for the diagnosis and treatment of BC.

A novel Lnc408 maintains breast cancer stem cell stemness by recruiting SP3 to suppress CBY1 transcription and increasing nuclear ß-catenin levels.

Tumor initiation, development, and relapse may be closely associated with cancer stem cells (CSCs). The complicated mechanisms underlying the maintenance of CSCs are keeping in illustration. Long noncoding RNAs (lncRNAs), due to their multifunction in various biological processes, have been indicated to play a crucial role in CSC renewal and stemness maintenance. Using lncRNA array, we identified a novel lncRNA (named lnc408) in epithelial-mesenchymal transition-related breast CSCs (BCSCs). The lnc408 is high expressed in BCSCs in vitro and in vivo. The enhanced lnc408 is critical to BCSC characteristics and tumorigenesis. Lnc408 can recruit transcript factor SP3 to CBY1 promoter to serve as an inhibitor in CBY1 transcription in BCSCs. The high expressed CBY1 in non-BCSC interacts with 14-3-3 and ß-catenin to form a ternary complex, which leads a translocation of the ternary complex into cytoplasm from nucleus and degradation of ß-catenin in phosphorylation-dependent pattern. The lnc408-mediated decrease of CBY1 in BCSCs impairs the formation of 14-3-3/ß-catenin/CBY1 complex, and keeps ß-catenin in nucleus to promote CSC-associated CD44, SOX2, Nanog, Klf4, and c-Myc expressions and contributes to mammosphere formation; however, restoration of CBY1 expression in tumor cells reduces BCSC and its enrichment, thus lnc408 plays an essential role in maintenance of BCSC stemness. In shortly, these findings highlight that the novel lnc408 functions as an oncogenic factor by recruiting SP3 to inhibit CBY1 expression and ß-catenin accumulation in nucleus to maintain stemness properties of BCSCs. Lnc408-CBY1-ß-catenin signaling axis might serve as a new diagnostic and therapeutic target for breast cancer.

SLC6A8-mediated intracellular creatine accumulation enhances hypoxic breast cancer cell survival via ameliorating oxidative stress.

BACKGROUND: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, with poor prognosis and limited treatment options. Hypoxia is a key hallmark of TNBC. Metabolic adaptation promotes progression of TNBC cells that are located within the hypoxic tumor regions. However, it is not well understood regarding the precise molecular mechanisms underlying the regulation of metabolic adaptions by hypoxia. METHODS: RNA sequencing was performed to analyze the gene expression profiles in MDA-MB-231 cell line (20% O2 and 1% O2). Expressions of Slc6a8, which encodes the creatine transporter protein, were detected in breast cancer cells and tissues by quantitative real-time PCR. Immunohistochemistry was performed to detect SLC6A8 protein abundances in tumor tissues. Clinicopathologic correlation and overall survival were evaluated by chi-square test and Kaplan-Meier analysis, respectively. Cell viability assay and flow cytometry analysis with Annexin V/PI double staining were performed to investigate the impact of SLC6A8-mediated uptake of creatine on viability of hypoxic TNBC cells. TNBC orthotopic mouse model was used to evaluate the effects of creatine in vivo. RESULTS: SLC6A8 was aberrantly upregulated in TNBC cells in hypoxia. SLC6A8 was drastically overexpressed in TNBC tissues and its level was tightly associated with advanced TNM stage, higher histological grade and worse overall survival of TNBC patients. We found that SLC6A8 was transcriptionally upregulated by p65/NF-κB and mediated accumulation of intracellular creatine in hypoxia. SLC6A8-mediated accumulation of creatine promoted survival and suppressed apoptosis via maintaining redox homeostasis in hypoxic TNBC cells. Furthermore, creatine was required to facilitate tumor growth in xenograft mouse models. Mechanistically, intracellular creatine bolstered cell antioxidant defense by reducing mitochondrial activity and oxygen consumption rates to reduce accumulation of intracellular reactive oxygen species, ultimately activating AKT-ERK signaling, the activation of which protected the viability of hypoxic TNBC cells via mediating the upregulation of Ki-67 and Bcl-2, and the downregulation of Bax and cleaved Caspase-3. CONCLUSIONS: Our study indicates that SLC6A8-mediated creatine accumulation plays an important role in promoting TNBC progression, and may provide a potential therapeutic strategy option for treatment of SLC6A8 high expressed TNBC.

Mirtronic miR-4646-5p promotes gastric cancer metastasis by regulating ABHD16A and metabolite lysophosphatidylserines.

The aberrant classical miRNAs are considered to play significant roles in tumor progression. However, it remains unclear for nonclassical miRNAs, a set of Drosha-independent miRNAs in the process of various biology. Here, we reveal that a nonclassical miR-4646-5p plays a pivotal role in gastric cancer (GC) metastasis. MiR-4646-5p, one of Drosha-independent mirtronic miRNA, is aberrant up-regulated in Drosha-low expressed GC and Drosha-knockdown gastric cancer cells. Mirtronic miR-4646-5p is a specific transcription splicing product of intron 3 of the host gene Abhd16a with the aid of SRSF2. The enhanced miR-4646-5p can stabilize HIF1A by targeting PHD3 to positive feedback regulate Abhd16a and miR-4646-5p itself expressions. ABHD16A, as an emerging phosphatidylserine-specific lipase, involves in lipid metabolism leading to lysophosphatidylserines (lyso-PSs) accumulation, which stimulates RhoA and downstream LIMK/cofilin cascade activity through GPR34/Gi subunit, thus causes metastasis of gastric cancer. In addition, miR-4646-5p/PHD3/HIF1A signaling can also up-regulate RhoA expression and synergistically promote gastric cancer cell invasion and metastasis. Our study provides new insights of nonclassical mirtronic miRNA on tumor progress and may serve as a new diagnostic biomarker for gastric cancer. MiR-4646-5p and its host gene Abhd16a mediated abnormal lipid metabolism may be a new target for clinical treatment of gastric cancer.

GPER-Induced ERK Signaling Decreases Cell Viability of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is an aggressive malignancy with a poor prognosis. Effective biomarkers and specific therapeutic targets for HCC are therefore urgently needed. G protein-coupled estrogen receptor (GPER) plays a crucial role in numerous cancer types; however, its functions in HCC require further exploration. In the present study, we found a remarkable difference in GPER staining between tumor tissue (100/141, 70.9%) and matched non-tumor tissue (27/30, 90.0%). Compared with the GPER-negative patients, the GPER-positive patients with HCC were closely associated with female sex, negative hepatitis B surface antigen, small tumor size, low serum alpha fetoprotein level, and longer overall survival. Treatment with GPER-specific agonist G1 led to the sustained and transient activation of the EGFR/ERK and EGFR/AKT signaling pathways, respectively, in the HCC cell lines HCCLM3 and SMMC-7721, which express high levels of GPER. Interestingly, G1-induced EGFR/ERK signaling, rather than EGFR/AKT signaling mediated by GPER, was involved in decreasing cell viability by blocking cell cycle progression, thereby promoting apoptosis and inhibiting cell growth. Clinical analysis indicated that simultaneous high expression of GPER and phosphorylated-ERK (p-ERK) predicted improved prognosis for HCC. Finally, the activation of GPER/ERK signaling remarkably suppressed tumor growth in an HCC xenograft model, and this result was consistent with the in vitro data. Our findings suggest that specific activation of the GPER/ERK axis may serve as a novel tumor-suppressive mechanism and that this axis could be a therapeutic target for HCC.