Reactivating PTEN to impair glioma stem cells by inhibiting cytosolic iron-sulfur assembly.

Glioblastoma, the most lethal primary brain tumor, harbors glioma stem cells (GSCs) that not only initiate and maintain malignant phenotypes but also enhance therapeutic resistance. Although frequently mutated in glioblastomas, the function and regulation of PTEN in PTEN-intact GSCs are unknown. Here, we found that PTEN directly interacted with MMS19 and competitively disrupted MMS19-based cytosolic iron-sulfur (Fe-S) cluster assembly (CIA) machinery in differentiated glioma cells. PTEN was specifically succinated at cysteine (C) 211 in GSCs compared with matched differentiated glioma cells. Isotope tracing coupled with mass spectrometry analysis confirmed that fumarate, generated by adenylosuccinate lyase (ADSL) in the de novo purine synthesis pathway that is highly activated in GSCs, promoted PTEN C211 succination. This modification abrogated the interaction between PTEN and MMS19, reactivating the CIA machinery pathway in GSCs. Functionally, inhibiting PTEN C211 succination by reexpressing a PTEN C211S mutant, depleting ADSL by shRNAs, or consuming fumarate by the US Food and Drug Administration-approved prescription drug N-acetylcysteine (NAC) impaired GSC maintenance. Reexpressing PTEN C211S or treating with NAC sensitized GSC-derived brain tumors to temozolomide and irradiation, the standard-of-care treatments for patients with glioblastoma, by slowing CIA machinery-mediated DNA damage repair. These findings reveal an immediately practicable strategy to target GSCs to treat glioblastoma by combination therapy with repurposed NAC.

Inhibiting G6PD by quercetin promotes degradation of EGFR T790M mutation.

EGFRT790M mutation causes resistance to the first-generation tyrosine kinase inhibitors (TKIs) in patients with non-small cell lung cancer (NSCLC). However, the therapeutic options for sensitizing first TKIs and delaying the emergence of EGFRT790M mutant are limited. In this study, we show that quercetin directly binds with glucose-6-phosphate dehydrogenase (G6PD) and inhibits its enzymatic activity through competitively abrogating NADP+ binding in the catalytic domain. This inhibition subsequently reduces intracellular NADPH levels, resulting in insufficient substrate for methionine reductase A (MsrA) to reduce M790 oxidization of EGFRT790M and inducing the degradation of EGFRT790M. Quercetin synergistically enhances the therapeutic effect of gefitinib on EGFRT790M-harboring NSCLCs and delays the acquisition of the EGFRT790M mutation. Notably, high levels of G6PD expression are correlated with poor prognosis and the emerging time of EGFRT790M mutation in patients with NSCLC. These findings highlight the potential implication of quercetin in overcoming EGFRT790M-driven TKI resistance by directly targeting G6PD.

Hypoxanthine phosphoribosyl transferase 1 metabolizes temozolomide to activate AMPK for driving chemoresistance of glioblastomas.

Temozolomide (TMZ) is a standard treatment for glioblastoma (GBM) patients. However, TMZ has moderate therapeutic effects due to chemoresistance of GBM cells through less clarified mechanisms. Here, we demonstrate that TMZ-derived 5-aminoimidazole-4-carboxamide (AICA) is converted to AICA ribosyl-5-phosphate (AICAR) in GBM cells. This conversion is catalyzed by hypoxanthine phosphoribosyl transferase 1 (HPRT1), which is highly expressed in human GBMs. As the bona fide activator of AMP-activated protein kinase (AMPK), TMZ-derived AICAR activates AMPK to phosphorylate threonine 52 (T52) of RRM1, the catalytic subunit of ribonucleotide reductase (RNR), leading to RNR activation and increased production of dNTPs to fuel the repairment of TMZ-induced-DNA damage. RRM1 T52A expression, genetic interruption of HPRT1-mediated AICAR production, or administration of 6-mercaptopurine (6-MP), a clinically approved inhibitor of HPRT1, blocks TMZ-induced AMPK activation and sensitizes brain tumor cells to TMZ treatment in mice. In addition, HPRT1 expression levels are positively correlated with poor prognosis in GBM patients who received TMZ treatment. These results uncover a critical bifunctional role of TMZ in GBM treatment that leads to chemoresistance. Our findings underscore the potential of combined administration of clinically available 6-MP to overcome TMZ chemoresistance and improve GBM treatment.

PGC1α Degradation Suppresses Mitochondrial Biogenesis to Confer Radiation Resistance in Glioma.

Radiotherapy is a major component of standard-of-care treatment for gliomas, the most prevalent type of brain tumor. However, resistance to radiotherapy remains a major concern. Identification of mechanisms governing radioresistance in gliomas could reveal improved therapeutic strategies for treating patients. Here, we report that mitochondrial metabolic pathways are suppressed in radioresistant gliomas through integrated analyses of transcriptomic data from glioma specimens and cell lines. Decreased expression of peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC1α), the key regulator of mitochondrial biogenesis and metabolism, correlated with glioma recurrence and predicted poor prognosis and response to radiotherapy of patients with glioma. The subpopulation of glioma cells with low-mitochondrial-mass exhibited reduced expression of PGC1α and enhanced resistance to radiotherapy treatment. Mechanistically, PGC1α was phosphorylated at serine (S) 636 by DNA-dependent protein kinase in response to irradiation. Phosphorylation at S636 promoted the degradation of PGC1α by facilitating its binding to the E3 ligase RNF34. Restoring PGC1α activity with expression of PGC1α S636A, a phosphorylation-resistant mutant, or a small-molecule PGC1α activator ZLN005 increased radiosensitivity of resistant glioma cells by reactivating mitochondria-related reactive oxygen species production and inducing apoptotic effects both in vitro and in vivo. In summary, this study identified a self-protective mechanism in glioma cells in which radiotherapy-induced degradation of PGC1α and suppression of mitochondrial biogenesis play a central role. Targeted activation of PGC1α could help improve response to radiotherapy in patients with glioma. SIGNIFICANCE: Glioma cells reduce mitochondrial biogenesis by promoting PGC1α degradation to promote resistance to radiotherapy, indicating potential therapeutic strategies to enhance radiosensitivity.

Chromium (VI)-induced ALDH1A1/EGF axis promotes lung cancer progression.

Cr(VI) is broadly applied in industry. Cr(VI) exposure places a big burden on public health, thereby increasing the risk of lung squamous cell carcinoma (LUSC). The mechanisms underlying Cr(VI)-induced LUSC remain largely elusive. Here, we report that the cancer stem cell (CSC)/tumour-initiating cell (TIC)-like subgroup within Cr(VI)-transformed bronchial epithelial cells (CrT) promotes lung cancer tumourigenesis. Mechanistically, Cr(VI) exposure specifically increases the expression levels of aldehyde dehydrogenase 1A1 (ALDH1A1), a CSC marker, through KLF4-mediated transcription. ALDH1A1 maintains self-renewal of CrT/TICs and facilitates the expression and secretion of EGF from CrT/TICs, which subsequently promotes the activation of EGFR signalling in differentiated cancer cells and tumour growth of LUSC. In addition, the ALDH1A1 inhibitor A37 and gemcitabine synergistically suppress LUSC progression. Importantly, high ALDH1A1 expression levels are positively correlated with advanced clinical stages and predict poor survival in LUSC patients. These findings elucidate how ALDH1A1 modulates EGF secretion from TICs to facilitate LUSC tumourigenesis, highlighting new therapeutic strategies for malignant lung cancers.

Diallyl trisulfide sensitizes radiation therapy on glioblastoma through directly targeting thioredoxin 1.

Radiotherapy is a standard-of-care treatment approach for glioblastoma (GBM) patients, but therapeutic resistance to radiotherapy remains a major challenge. Here we demonstrate that diallyl trisulfide (DATS) directly conjugates with cysteine (C) 32 and C35 (C32/35) residues of thioredoxin 1 (Trx1) through Michael addition reactions. Due to localizing in activity center of Trx1, the conjugation between DATS and C32/35 results in inhibition of Trx1 activity, therefore disturbing thioredoxin system and leading to accumulated levels of reactive oxygen species (ROS). High levels of Trx1 expression are correlated with poor prognosis of glioma patients. Notably, we reveal that DATS synergistically enhances irradiation (IR)-induced ROS accumulation, apoptosis, DNA damage, as well as inhibition of tumor growth of GBM cells. These findings highlight the potential benefits of DATS in sensitizing radiotherapy of GBM patients.

Benzo[a]pyrene-decreased gap junctional intercellular communication via calcium/calmodulin signaling increases apoptosis in TM4 cells.

The mechanism of male reproductive toxicity induced by benzo[a]pyrene (BaP) is poorly understood. Gap junctional intercellular communication (GJIC) is known to play a critical role in maintaining spermatogenesis. The aim of the present study was to determine the toxic effects of BaP in Sertoli cells, and to explore the possibility and potential mechanisms of BaP-induced changes in the level of GJIC, and the relationship between GJIC and BaP-induced apoptosis. We treated mouse Sertoli cell lines (TM4) with different concentrations (0.1-100 µm) of BaP for 1-48 hours, and found that GJIC exhibited a dose- and time-dependent downregulation. Treatment with 10 µm BaP increased apoptosis, intracellular Ca2+ level ([Ca2+ ]i ) and calmodulin (CaM) protein expression, and decreased the protein level of connexin 43 (Cx43) (also known as gap junction α-1 protein [GJA1]) in TM4 cells. However, BaP had no effect on the phosphorylation of Cx43 at Ser279/282, Ser255, Ser368 or Ser262. Downregulation of [Ca2+ ]i by BAPTA-AM significantly attenuated the BaP-induced GJIC suppression, Cx43 protein decrease and CaM protein increase. Interestingly, inhibition of CaM expression by W7 partially recovered BaP-induced GJIC inhibition, but had no effect on BaP-induced Cx43 protein decrease. Pretreatment with the GJIC activator retinoic acid significantly mitigated BaP-induced apoptosis. In conclusion, these results suggest that BaP can decrease GJIC via Ca2+ /CaM signaling, and that BaP-induced GJIC suppression increases apoptosis in TM4 cells.

Gap junctional intercellular communication and endoplasmic reticulum stress regulate chronic cadmium exposure induced apoptosis in HK-2 cells.

Cadmium (Cd), a toxic heavy metal, is known to induce renal toxicity by primarily targeting at renal proximal tubule. Endoplasmic reticulum (ER) stress and gap junctional intercellular communication (GJIC) regulate many pathophysiological processes. Yet, how ER stress and GJIC regulate Cd-induced nephrotoxicity remain elusive. In this study, we treated human proximal tubule (HK-2) cells with 1 µM CdCl2 every other day for 12 days and found that Cd significantly increased cell apoptosis at 10 and 12 days. This cytotoxicity correlated with activation of ER stress and apoptotic signaling evidenced by upregulation of inositol-requiring enzyme 1 (IRE1α), splice X-box binding protein-1 (XBP-1s), and apoptosis signal-regulating kinase 1 (ASK1) proteins. Interestingly, the AKT signaling was activated at 2- and 4-day and then inhibited at 10- and 12-day of Cd treatment; by contrast, Cd decreased GJIC levels at 2- and 4-day followed by a significant increase at 10- and 12-day treatment. Activation of AKT by SC79 or inhibition of GJIC by 18α-glycyrrhetinic acid (18α-GA) completely abolished Cd-induced AKT inhibition and IRE1α-ASK1 activation. Importantly, pretreatment with ER stress inhibitor or 18α-GA significantly mitigated Cd-induced apoptosis. These results suggest that GJIC collaborates with AKT signaling and ER stress in regulating prolonged Cd-treatment-induced apoptosis in HK-2 cells.

Connexin 43 mediates changes in protein phosphorylation in HK-2 cells during chronic cadmium exposure.

Connexin 43 (Cx43) is believed to play a role in the mechanisms of toxicity of many chemical species, include cadmium (Cd). In this study, human renal proximal tubule (HK-2) cells were exposed to Cd (1µM, 10 days). Of the 584 protein residues detected using a Phospho Explorer antibody microarray (PEX100), more than half changed their levels of phosphorylation after chronic Cd exposure. Cx43 siRNA attenuated Cd-induced apoptosis and inhibited proliferation, while also attenuating changes in the levels of phosphorylation of many protein residues. According to DAVID Bioinformatics Resources analysis and KEGG PATHWAY database, AKT signal pathway may be the important one. Focusing on the AKT pathway confirmed that Cx43 mediated increased levels of p-PTENSer380/Ser382/Thr383 and decreased levels of p-AKTThr308, p-AKTTyr326, p-ASK1Ser83, and p-p27Thr187, thereby possibly contributing to the Cd-induced apoptosis and inhibited proliferation. These results suggested that AKT pathway was the dominant pathway involved in Cx43-mediated chronic Cd toxicity.

Role of connexin 43 in cadmium-induced proliferation of human prostate epithelial cells.

Connexins (Cxs), the subunits of gap junction channels, are involved in many physiological processes. Aberrant control of Cxs and gap junction intercellular communication may contribute to many diseases, including the promotion of cancer. Cd exposure is associated with increased risk of human prostate cancer and benign prostatic hyperplasia. The roles of Cxs in the effects of Cd on the prostate have, however, not been reported previously. In this study, the human prostate epithelial cell line RWPE-1 was exposed to Cd. A low dose of Cd stimulated cell proliferation along with a lower degree of gap junction intercellular communication and an elevated level of the protein Cx43. Cd exposure increased the levels of intracellular Ca2+ and phosphorylated Cx43 at the Ser368 site. Knockdown of Cx43 using siRNA blocked Cd-induced proliferation and interfered with the Cd-induced changes in the protein levels of cyclin D1, cyclin B1, p27Kip1 (p27) and p21Waf1/Cip1 (p21). The increase in Cx43 expression induced by Cd was presumably mediated by the androgen receptor, because it was abolished upon treatment with the androgen receptor antagonist, flutamide. Thus, a low dose of Cd promotes cell proliferation in RWPE-1, possibly mediated by Cx43 expression through an effect on cell cycle-associated proteins. Cx43 might be a target for prostatic diseases associated with Cd exposure. Copyright © 2017 John Wiley & Sons, Ltd.