Reversal of multidrug resistance by stimuli-responsive drug delivery systems for therapy of tumor.

Intrinsic resistance to cytotoxic drugs has been a main issue in cancer therapy for decades. Microenvironmental acidity is a simple while highly efficient mechanism of chemoresistance, exploited through impairment of drug delivery. The latter is achieved by extracellular protonation and/or sequestration into acidic vesicles. This study investigates the importance of extracellular acidosis and nanovesicle (exosome) release in the resistance of human tumour cell to cisplatin (CisPt); in parallel to proton pump inhibitors (PPI) ability of interfering with these tumour cell features. The results showed that CisPt uptake by human tumour cells was markedly impaired by low pH conditions. Moreover, exosomes purified from supernatants of these cell cultures contained various amounts of CisPt, which correlated to the pH conditions of the culture medium. HPLC-Q-ICP-MS analysis revealed that exosome purified from tumour cell culture supernatants contained CisPt in its native form. PPI pre-treatment increased cellular uptake of CisPt, as compared to untreated cells, in an acidic-depend manner. Furthermore, it induced a clear inhibition of exosome release by tumour cells. Human tumours obtained from xenografts pretreated with PPI contained more CisPt as compared to tumours from xenografts treated with CisPt alone. Further analysis showed that in vivo PPI treatment induced a clear reduction in the plasmatic levels of tumour-derived exosomes which also contained lower level of CisPt. Altogether, these findings point to the identification of a double mechanism that human malignant melanoma use in resisting to a dreadful cellular poison such as cisplatin. This framework of resistance includes both low pH-dependent extracellular sequestration and an exosome-mediated elimination. Both mechanisms are markedly impaired by proton pump inhibition, leading to an increased CisPt-dependent cytotoxicity.

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Exosome Release and Low pH Belong to a Framework of Resistance of Human Melanoma Cells to Cisplatin

The vacuolar ATPases (V-ATPases) are a family of proton pumps that couple ATP hydrolysis to proton transport into intracellular compartments and across the plasma membrane. They function in a wide ...

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The Function of V-ATPases in Cancer

Ceramide synthases as potential targets for therapeutic intervention in human diseases

Acidity is a hallmark of malignant tumor, representing a very efficient mechanism of chemoresistance. Proton pump inhibitors (PPI) at high dosage have been shown to sensitize chemoresistant human tumor cells and tumors to cytotoxic molecules. The aim of this pilot study was to investigate the efficacy of PPI in improving the clinical outcome of docetaxel + cisplatin regimen in patients with metastatic breast cancer (MBC). Patients enrolled were randomly assigned to three arms: Arm A, docetaxel 75 mg/m2 followed by cisplatin 75 mg/m2 on d4, repeated every 21 days with a maximum of 6 cycles; Arm B, the same chemotherapy preceded by three days esomeprazole (ESOM) 80 mg p.o. bid, beginning on d1 repeated weekly. Weekly intermittent administration of ESOM (3 days on 4 days off) was maintained up to maximum 66 weeks; Arm C, the same as Arm B with the only difference being dose of ESOM at 100 mg p.o. bid. The primary endpoint was response rate. Ninety-four patients were randomly assigned and underwent at least one injection of chemotherapy. Response rates for arm A, B and C were 46.9, 71.0, and 64.5 %, respectively. Median TTP for arm A (n = 32), B (n = 31), C (n = 31) were 8.7, 9.4, and 9.7 months, respectively. A significant difference was observed between patients who had taken PPI and who not with ORR (67.7 % vs. 46.9 %, p = 0.049) and median TTP (9.7 months vs. 8.7 months, p = 0.045). Exploratory analysis showed that among 15 patients with triple negative breast cancer (TNBC), this difference was bigger with median TTP of 10.7 and 5.8 months, respectively (p = 0.011). PPI combination showed a marked effect on OS as well, while with a borderline significance (29.9 vs. 19.2 months, p = 0.090). No additional toxicity was observed with PPI. The results of this pilot clinical trial showed that intermittent high dose PPI enhance the antitumor effects of chemotherapy in MBC patients without evidence of additional toxicity, which requires urgent validation in a multicenter, randomized, phase III trial. Clinicaltrials.gov identifier: NCT01069081
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Intermittent high dose proton pump inhibitor enhances the antitumor effects of chemotherapy in metastatic breast cancer.

A main obstacle to overcome during the treatment of tumors is drug resistance to chemotherapy; emerging studies indicate that a key factor contributing to this problem is the acidic tumor microenvironment. Here, we found that LASS2 expression was significantly lower in drug-resistant Michigan Cancer Foundation-7/adriamycin (MCF-7/ADR) human breast cancer cells than the drug-sensitive MCF-7 cells, and low expression of LASS2 was associated with poor prognosis in patients with breast cancer. Our results showed that the overexpression of LASS2 in MCF-7/ADR cells increased the chemosensitivity to multiple chemotherapeutic agents, including doxorubicin (Dox), whereas LASS2 knockdown in MCF-7 cells decreased the chemosensitivity. Cell-cycle analysis revealed a corresponding increase in apoptosis in the LASS2-overexpressing cells following Dox exposure, showing that the overexpression of LASS2 increased the susceptibility to Dox cytotoxicity. This effect was mediated by a significant increase in pHe (extracellular pH) and lysosomal pH, and more Dox entered the cells and stayed in the nuclei of cells. In nude mice, the combination of LASS2 overexpression and Dox significantly inhibited the growth of xenografts. Our findings suggest that LASS2 is involved in chemotherapeutic outcomes and low LASS2 expression may predict chemoresistance.

LASS2 enhances chemosensitivity of breast cancer by counteracting acidic tumor microenvironment through inhibiting activity of V-ATPase proton pump.

Dysregulation of Hippo/YAP signaling leads to aberrant cell growth and neoplasia. Although the roles and regulation of Hippo/YAP signaling were extensively studied in cancer biology recently, study systematically checking the expression pattern of core components of this pathway at the tumor tissue level remains lacking. In this study, we thoroughly examined the profile of core components of Hippo/YAP signaling in patient specimens both at the mRNA and at protein levels. We found that the mRNA level of YAP1/TAZ and their target genes, CRY61, CTGF, and BIRC5, was remarkably amplified in glioma tissues. Consistently, the protein level of YAP1/TAZ increased and meanwhile those of p-YAP1/p-TAZ and LATS1/2 decreased in gliomas. Unexpectedly, both the mRNA and protein levels of MST1/2 increased in the glioma tissues, inconsistent with its presumed tumor suppressor identity. In addition, over-expression of LATS2 decreased, while over-expression of YPA1 increased the cell proliferation ability. Furthermore, based on the data from the free public database, YAP1/TAZ and BIRC5 were positively associated with the prognosis of glioma patients, while LATS1/2 exhibited negative correlation with the glioma patient prognosis. Collectively, we deduce that, in glioma tissue context, MST1/2 may not be the essential component of the hippo/YAP pathway. Moreover, our findings uncover a new evidence supporting that YAP1/TAZ-BIRC5 might be abnormally activated due to LATS1/2 down-regulation, which in turn promote the occurrence and development of gliomas, paving the way to identify the potential therapeutic molecular target for gliomas.

Expression and significance of Hippo/YAP signaling in glioma progression.

Hippo/YAP pathway is known to be important for development, growth and organogenesis, and dysregulation of this pathway leads to tumor progression.We and others find that YAP is up-regulated in human gliomas and associated with worse prognosis of patients. However, the role and mechanism of YAP in glioma progression is largely unknown. The expression of YAP in glioma tissues was detected by quantitative polymerase chain reaction (qPCR) and immunoblotting. The effect of YAP on glioma progression was examined using cell growth assays and intracranial glioma model. The effect of YAP on β-catenin protein level, subcellular location and transcription activity was examined by immunoblotting, immunofluorescence and RT-PCR. Firstly, knockdown of YAP inhibited glioma cell proliferation in vitro and tumor growth in vivo. In addition, YAP modulated the protein level, subcellular location and transcription activity of β-catenin via regulating the activity of GSK3β. Lastly, β-catenin partially mediated the effect of YAP on glioma cell proliferation. Our findings identify that YAP promotes human glioma growth through enhancing Wnt/β-catenin signaling. In addition, this study provides a new crosstalk mechanism between Hippo/YAP and Wnt/β-catenin pathways, which suggests a new strategy for human glioma treatment.

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β-catenin-mediated YAP signaling promotes human glioma growth.

Mammalian sterile 20-like 1 (Mst1), an upstream serine/threonine-specific protein kinase of the Hippo pathway, is reported to play important roles in tumor suppression and organ size regulation in mammals via regulating cell proliferation and survival. However, whether it is involved in the pathogenesis of malignant gliomas remains poorly understood. Therefore, in the present work, we examined the effect and mechanism of Mst1 on the proliferation and apoptosis of malignant glioma cells. The cell proliferation and growth of glioma cells were examined by EdU incorporation and CCK-8 assay. In addition, the cell apoptosis was assessed by flow cytometry. We found that down-regulation of Mst1 promoted glioma cell proliferation and growth, but inhibited the cell apoptosis. Consistent with this, over-expression of Mst1 inhibited glioma cell proliferation and growth. Interestingly, Mst1 did not affect the phosphorylation of YAP1, the key downstream molecule of Hippo pathway. However, Mst1 was found to bind to AKT in glioma cell and negatively regulated AKT and mTOR activity. Finally, the increased cell proliferation rate induced by Mst1 down-regulation was partially abolished by down-regulation of AKT1. Meanwhile, glioma cell growth inhibition induced by Mst1 over-expression was partially rescued by over-expression of AKT1. Taken together, these findings suggest that Mst1 regulates proliferation of glioma cells via AKT/mTOR signaling pathway.

Mst1 regulates glioma cell proliferation via the AKT/mTOR signaling pathway

Previously, we have reported that Yes-associated protein (YAP) is upregulated in human glioma tissues and its level is positively correlated with patient prognosis. However, the role and mechanism of YAP in the highly invasive nature of human gliomas were largely unknown. In this study, examined by wound healing assay, transwell assay, or live-imaging, we found that YAP downregulation inhibited glioma cell migration and invasion, while YAP over-expression promoted them. Interestingly, the above effect of YAP on immortalized glioma cells was recapitulated in cultured primary glioma cells. In addition, the protein level of N-cadherin and Twist, two important proteins involved in tumor invasion, increased after YAP over-expression. Meanwhile, YAP over-expression significantly increased the F-actin level and changed the distribution of F-actin, leading to cytoskeletal reorganization, which plays an important role in cell motility. Furthermore, the promotion effect of YAP over-expression on glioma cell migration and invasion was partially abolished by Twist downregulation. Taken together, our findings show that YAP contributes to glioma cell migration and invasion by regulating N-cadherin and Twist, as well as cytoskeletal reorganization.

Yan Wang

Papers

LASS2 enhances chemosensitivity of breast cancer by counteracting acidic tumor microenvironment through inhibiting activity of V-ATPase proton pump.

Expression and significance of Hippo/YAP signaling in glioma progression.

β-catenin-mediated YAP signaling promotes human glioma growth.

Mst1 regulates glioma cell proliferation via the AKT/mTOR signaling pathway

YAP Promotes Migration and Invasion of Human Glioma Cells.