Dual Role of CXCL8 in Maintaining the Mesenchymal State of Glioblastoma Stem Cells and M2-Like Tumor-Associated Macrophages.

PURPOSE: The dynamic interplay between glioblastoma stem cells (GSC) and tumor-associated macrophages (TAM) sculpts the tumor immune microenvironment (TIME) and promotes malignant progression of glioblastoma (GBM). However, the mechanisms underlying this interaction are still incompletely understood. Here, we investigate the role of CXCL8 in the maintenance of the mesenchymal state of GSC populations and reprogramming the TIME to an immunosuppressive state. EXPERIMENTAL DESIGN: We performed an integrative multi-omics analyses of RNA sequencing, GBM mRNA expression datasets, immune signatures, and epigenetic profiling to define the specific genes expressed in the mesenchymal GSC subsets. We then used patient-derived GSCs and a xenograft murine model to investigate the mechanisms of tumor-intrinsic and extrinsic factor to maintain the mesenchymal state of GSCs and induce TAM polarization. RESULTS: We identified that CXCL8 was preferentially expressed and secreted by mesenchymal GSCs and activated PI3K/AKT and NF-κB signaling to maintain GSC proliferation, survival, and self-renewal through a cell-intrinsic mechanism. CXCL8 induced signaling through a CXCR2-JAK2/STAT3 axis in TAMs, which supported an M2-like TAM phenotype through a paracrine, cell-extrinsic pathway. Genetic- and small molecule-based inhibition of these dual complementary signaling cascades in GSCs and TAMs suppressed GBM tumor growth and prolonged survival of orthotopic xenograft-bearing mice. CONCLUSIONS: CXCL8 plays critical roles in maintaining the mesenchymal state of GSCs and M2-like TAM polarization in GBM, highlighting an interplay between cell-autonomous and cell-extrinsic mechanisms. Targeting CXCL8 and its downstream effectors may effectively improve GBM treatment.

GAPDH facilitates homologous recombination repair by stabilizing RAD51 in an HDAC1-dependent manner.

Homologous recombination (HR), a form of error-free DNA double-strand break (DSB) repair, is important for the maintenance of genomic integrity. Here, we identify a moonlighting protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as a regulator of HR repair, which is mediated through HDAC1-dependent regulation of RAD51 stability. Mechanistically, in response to DSBs, Src signaling is activated and mediates GAPDH nuclear translocation. Then, GAPDH directly binds with HDAC1, releasing it from its suppressor. Subsequently, activated HDAC1 deacetylates RAD51 and prevents it from undergoing proteasomal degradation. GAPDH knockdown decreases RAD51 protein levels and inhibits HR, which is re-established by overexpression of HDAC1 but not SIRT1. Notably, K40 is an important acetylation site of RAD51, which facilitates stability maintenance. Collectively, our findings provide new insights into the importance of GAPDH in HR repair, in addition to its glycolytic activity, and they show that GAPDH stabilizes RAD51 by interacting with HDAC1 and promoting HDAC1 deacetylation of RAD51.

Excessive iron inhibits insulin secretion via perturbing transcriptional regulation of SYT7 by OGG1.

Although iron overload is closely related to the occurrence of type 2 diabetes mellitus (T2DM), the specific mechanism is unclear. Here, we found that excessive iron inhibited the secretion of insulin (INS) and impaired islet ß cell function through downregulating Synaptotagmin 7 (SYT7) in iron overload model in vivo and in vitro. Our results further demonstrated that 8-oxoguanine DNA glycosylase (OGG1), a key protein in the DNA base excision repair, was an upstream regulator of SYT7. Interestingly, such regulation could be suppressed by excessive iron. Ogg1-null mice, iron overload mice and db/db mice exhibit reduced INS secretion, weakened ß cell function and subsequently impaired glucose tolerance. Notably, SYT7 overexpression could rescue these phenotypes. Our data revealed an intrinsic mechanism by which excessive iron inhibits INS secretion through perturbing the transcriptional regulation of SYT7 by OGG1, which suggested that SYT7 was a potential target in clinical therapy for T2DM.

Sterol regulatory element-binding protein 2 maintains glioblastoma stem cells by keeping the balance between cholesterol biosynthesis and uptake.

BACKGROUND: Glioblastomas (GBMs) display striking dysregulation of metabolism to promote tumor growth. Glioblastoma stem cells (GSCs) adapt to regions of heterogeneous nutrient availability, yet display dependency on de novo cholesterol biosynthesis. The transcription factor Sterol Regulatory Element-Binding Protein 2 (SREBP2) regulates cholesterol biosynthesis enzymes and uptake receptors. Here, we investigate adaptive behavior of GSCs under different cholesterol supplies. METHODS: In silico analysis of patient tumors demonstrated enrichment of cholesterol synthesis associated with decreased angiogenesis. Comparative gene expression of cholesterol biosynthesis enzymes in paired GBM specimens and GSCs were performed. In vitro and in vivo loss-of-function genetic and pharmacologic assays were conducted to evaluate the effect of SREBP2 on GBM cholesterol biosynthesis, proliferation, and self-renewal. Chromatin immunoprecipitation quantitative real-time PCR was leveraged to map the regulation of SREBP2 to cholesterol biosynthesis enzymes and uptake receptors in GSCs. RESULTS: Cholesterol biosynthetic enzymes were expressed at higher levels in GBM tumor cores than in invasive margins. SREBP2 promoted cholesterol biosynthesis in GSCs, especially under starvation, as well as proliferation, self-renewal, and tumor growth. SREBP2 governed the balance between cholesterol biosynthesis and uptake in different nutrient conditions. CONCLUSIONS: SREBP2 displays context-specific regulation of cholesterol biology based on its availability in the microenvironment with induction of cholesterol biosynthesis in the tumor core and uptake in the margin, informing a novel treatment strategy for GBM.

ß2-Microglobulin Maintains Glioblastoma Stem Cells and Induces M2-like Polarization of Tumor-Associated Macrophages.

Glioblastoma (GBM) is a complex ecosystem that includes a heterogeneous tumor population and the tumor-immune microenvironment (TIME), prominently containing tumor-associated macrophages (TAM) and microglia. Here, we demonstrated that ß2-microglobulin (B2M), a subunit of the class I major histocompatibility complex (MHC-I), promotes the maintenance of stem-like neoplastic populations and reprograms the TIME to an anti-inflammatory, tumor-promoting state. B2M activated PI3K/AKT/mTOR signaling by interacting with PIP5K1A in GBM stem cells (GSC) and promoting MYC-induced secretion of transforming growth factor-ß1 (TGFß1). Inhibition of B2M attenuated GSC survival, self-renewal, and tumor growth. B2M-induced TGFß1 secretion activated paracrine SMAD and PI3K/AKT signaling in TAMs and promoted an M2-like macrophage phenotype. These findings reveal tumor-promoting functions of B2M and suggest that targeting B2M or its downstream axis may provide an effective approach for treating GBM. SIGNIFICANCE: ß2-microglobulin signaling in glioblastoma cells activates a PI3K/AKT/MYC/TGFß1 axis that maintains stem cells and induces M2-like macrophage polarization, highlighting potential therapeutic strategies for targeting tumor cells and the immunosuppressive microenvironment in glioblastoma.

Arginine methylation of APE1 promotes its mitochondrial translocation to protect cells from oxidative damage.

Apurinic/apyrimidinic endonuclease 1 (APE1) is an essential multifunctional protein in mammals that plays critical roles in DNA repair and redox signaling within the cell. Impaired APE1 function or dysregulation is associated with disease susceptibility and poor cancer prognosis. Orchestrated regulatory mechanisms are crucial to ensure its function in a specific subcellular location at specific time. Here, we report arginine methylation as a post-translational modification (PTM) that regulates APE1 translocation to mitochondria in HeLa and HEK-293 cells. Protein arginine methyl-transferase 1 (PRMT1) was shown to methylate APE1 in vitro. Site-directed mutagenesis identified R301 as the major methylation site. We confirmed that APE1 is methylated in cells and that the R301K mutation significantly reduces its methylation. Baseline mitochondrial APE1 levels were low under standard culture conditions, but they could be induced by oxidative agents. Methylation-deficient APE1 showed reduced mitochondrial translocation. Methylation affected the interaction of APE1 with Tom20, translocase of the outer mitochondrial membrane. Methylation-deficient APE1 resulted in increased mitochondrial DNA damage and increased cytochrome c release after stimuli. These data suggest that methylation of APE1 promotes its mitochondrial translocation and protects cells from oxidative damage. This work describes a novel PTM regulation model of APE1 subcellular distribution through arginine methylation.

DNA polymerase beta modulates cancer progression via enhancing CDH13 expression by promoter demethylation.

DNA polymerase ß (Pol ß) plays a critical role in DNA base excision repair (BER), which is involved in maintaining genomic stability and in the modulation of DNA demethylation. Numerous studies implicated deficiency of Pol ß in the genomic instability and dysregulation of genes expression, leading to affecting initiation of cancer. However, the role of Pol ß in cancer progression is still unclear. Here, we show that Pol ß depresses migratory and invasive capabilities of both breast and lung carcinomas, which were evident in human breast and lung cancer cells, as well as in mouse xenograft tumors. On the molecular basis, overexpression of Pol ß enhanced expression of CDH13, which show function on cell adhesion and migration. Knockdown of CDH13 restores the migratory, invasive capabilities and angiogenesis in tumor, which gets impaired by Pol ß. According to the function of BER on modulation of DNA demethylation, our studies on CDH13 expression and the DNA methylation levels of CDH13 promoter suggested that Pol ß promotes expression of CDH13 by augmenting DNA demethylation of CDH13 promoter. Our findings elucidated a novel possibility that Pol ß impair cancer cell metastasis during cancer progression and shed light on the role of Pol ß in cancer therapy.

Src-mediated phosphorylation of GAPDH regulates its nuclear localization and cellular response to DNA damage.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme involved in energy metabolism. Recently, GAPDH has been suggested to have extraglycolytic functions in DNA repair, but the underlying mechanism for the GAPDH response to DNA damage remains unclear. Here, we demonstrate that the tyrosine kinase Src is activated under DNA damage stress and phosphorylates GAPDH at Tyr41. This phosphorylation of GAPDH is essential for its nuclear translocation and DNA repair function. Blocking the nuclear import of GAPDH by suppressing Src signaling or through a GAPDH Tyr41 mutation impairs its response to DNA damage. Nuclear GAPDH is recruited to DNA lesions and associates with DNA polymerase ß (Pol ß) to function in DNA repair. Nuclear GAPDH promotes Pol ß polymerase activity and increases base excision repair (BER) efficiency. Furthermore, GAPDH knockdown dramatically decreases BER efficiency and sensitizes cells to DNA damaging agents. Importantly, the knockdown of GAPDH in colon cancer SW480 cells and xenograft models effectively enhances their sensitivity to the chemotherapeutic drug 5-FU. In summary, our findings provide mechanistic insight into the new function of GAPDH in DNA repair and suggest a potential therapeutic target in chemotherapy.

Inhibition of AKT Sensitizes Cancer Cells to Antineoplastic Drugs by Downregulating Flap Endonuclease 1.

DNA repair mechanisms are crucial for cell survival. It increases the cancer cell's ability to resist DNA damage. FEN1 is involved in DNA replication and repair, specifically long-patch base excision repair. Although the gene function and post-translational modification of FEN1 are well studied, the regulatory mechanism of FEN1 by upstream signal pathways remains unclear. In this article, we have identified AKT as a regulator of FEN1 activity in lung cancer cells. Sustained activation of AKT can phosphorylate nuclear transcription factor NF-κB/p65. NF-κB/p65 directly binds to FEN1 promoter to promote a high transcription level of FEN1, revealing the contribution of the AKT signaling pathway to drug resistance of cancer cells. The combination of an AKT inhibitor and cisplatin efficiently suppressed lung cancer cell growth both in vitro and in vivo Our study illustrated an upstream regulatory mechanism of FEN1, which will contribute to the development of effective lung cancer therapies.These findings identified AKT as a regulator of FEN1 activity and revealed the AKT signaling pathway's contribution to drug resistance, which will contribute to the development of effective lung cancer therapy.

Two-way crosstalk between BER and c-NHEJ repair pathway is mediated by Pol-ß and Ku70.

Multiple DNA repair pathways may be involved in the removal of the same DNA lesion caused by endogenous or exogenous agents. Although distinct DNA repair machinery fulfill overlapping roles in the repair of DNA lesions, the mechanisms coordinating different pathways have not been investigated in detail. Here, we show that Ku70, a core protein of nonhomologous end-joining (NHEJ) repair pathway, can directly interact with DNA polymerase-ß (Pol-ß), a central player in the DNA base excision repair (BER), and this physical complex not only promotes the polymerase activity of Pol-ß and BER efficiency but also enhances the classic NHEJ repair. Moreover, we find that DNA damages caused by methyl methanesulfonate (MMS) or etoposide promote the formation of Ku70-Pol-ß complexes at the repair foci. Furthermore, suppression of endogenous Ku70 expression by small interfering RNA reduces BER efficiency and leads to higher sensitivity to MMS and accumulation of the DNA strand breaks. Similarly, Pol-ß knockdown impairs total-NHEJ capacity but only has a slight influence on alternative NHEJ. These results suggest that Pol-ß and Ku70 coordinate 2-way crosstalk between the BER and NHEJ pathways.-Xia, W., Ci, S., Li, M., Wang, M., Dianov, G. L., Ma, Z., Li, L., Hua, K., Alagamuthu, K. K., Qing, L., Luo, L., Edick, A. M., Liu, L., Hu, Z., He, L., Pan, F., Guo, Z. Two-way crosstalk between BER and c-NHEJ repair pathway is mediated by Pol-ß and Ku70.