Sunitinib, a multitargeted receptor tyrosine kinase inhibitor including vascular endothelial growth factor, has been widely used as a first‐line treatment against metastatic renal cell carcinoma ...(mRCC). However, mRCC often acquires resistance to sunitinib, rendering it difficult to treat with this agent. Recently, Rapalink‐1, a drug that links rapamycin and the mTOR kinase inhibitor MLN0128, has been developed with excellent therapeutic effects against breast cancer cells carrying mTOR resistance mutations. The aim of the present study was to evaluate the in vitro and in vivo therapeutic efficacy of Rapalink‐1 against renal cell carcinoma (RCC) compared to temsirolimus, which is commonly used as a small molecule inhibitor of mTOR and is a derivative of rapamycin. In comparison with temsirolimus, Rapalink‐1 showed significantly greater effects against proliferation, migration, invasion and cFolony formation in sunitinib‐naïve RCC cells. Inhibition was achieved through suppression of the phosphorylation of substrates in the mTOR signal pathway, such as p70S6K, eukaryotic translation initiation factor 4E‐binding protein 1 (4EBP1) and AKT. In addition, Rapalink‐1 had greater tumor suppressive effects than temsirolimus against the sunitinib‐resistant 786‐o cell line (SU‐R 786‐o), which we had previously established, as well as 3 additional SU‐R cell lines established here. RNA sequencing showed that Rapalink‐1 suppressed not only the mTOR signaling pathway but also a part of the MAPK signaling pathway, the ErbB signaling pathway and ABC transporters that were associated with resistance to several drugs. Our study suggests the possibility of a new treatment option for patients with RCC that is either sunitinib‐sensitive or sunitinib‐resistant.
A new mTOR inhibitor, Rapalink‐1, suppresses the MAPK signaling pathway, the ErbB signaling pathway and ABC transporters.
Continuous activation of hypoxia-inducible factor (HIF) is important for progression of renal cell carcinoma (RCC) and acquired resistance to antiangiogenic multikinase and mTOR inhibitors. Recently, ...HIF2α antagonists PT2385 and PT2399 were developed and are being evaluated in a phase I clinical trial for advanced or metastatic clear cell RCC (ccRCC). However, resistance to HIF2α antagonists would be expected to develop. In this study, we identified signals activated by HIF2α deficiency as candidate mediators of resistance to the HIF2α antagonists. We established sunitinib-resistant tumor cells
and created HIF2α-deficient variants of these cells using CRISPR/Cas9 technology. Mechanistic investigations revealed that a regulator of the serine biosynthesis pathway, phosphoglycerate dehydrogenase (PHGDH), was upregulated commonly in HIF2α-deficient tumor cells along with the serine biosynthesis pathway itself. Accordingly, treatment with a PHGDH inhibitor reduced the growth of HIF2α-deficient tumor cells
and
by inducing apoptosis. Our findings identify the serine biosynthesis pathway as a source of candidate therapeutic targets to eradicate advanced or metastatic ccRCC resistant to HIF2α antagonists.
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Sunitinib is the most common primary molecular-targeted agent for metastatic clear cell renal cell carcinoma (ccRCC); however, intrinsic or acquired sunitinib resistance has become a significant ...problem in medical practice. The present study focused on microRNA (miR)-99a-3p, which was significantly downregulated in clinical sunitinib-resistant ccRCC tissues in previous screening analyses, and investigated the molecular network associated with it. The expression levels of miR-99a-3p and its candidate target genes were evaluated in RCC cells, including previously established sunitinib-resistant 786-o (SU-R-786-o) cells, and clinical ccRCC tissues, using reverse transcription-quantitative polymerase chain reaction. Gain-of-function studies demonstrated that miR-99a-3p significantly suppressed cell proliferation and colony formation in RCC cells, including the SU-R-786-o cells, by inducing apoptosis. Based on in silico analyses and RNA sequencing data, followed by luciferase reporter assays, ribonucleotide reductase regulatory subunit-M2 (RRM2) was identified as a direct target of miR-99a-3p in the SU-R-786-o cells. Loss-of-function studies using small interfering RNA against RRM2 revealed that cell proliferation and colony growth were significantly inhibited via induction of apoptosis, particularly in the SU-R-786-o cells. Furthermore, the RRM2 inhibitor Didox (3,4-dihydroxybenzohydroxamic acid) exhibited anticancer effects in the SU-R-786-o cells and other RCC cells. To the best of our knowledge, this is the first report demonstrating that miR-99a-3p directly regulates RRM2. Identifying novel genes targeted by tumor-suppressive miR-99a-3p in sunitinib-resistant RCC cells may improve our understanding of intrinsic or acquired resistance and facilitate the development of novel therapeutic strategies.
The alcohol-abuse drug disulfiram has antitumor effects against diverse cancer types via inhibition of the ubiquitin-proteasome protein nuclear protein localization protein 4 (NPL4). However, the ...antitumor effects of NPL4 and disulfiram in clear cell renal cell carcinoma (ccRCC) are unclear. Here, we evaluated the therapeutic potential of targeting the ubiquitin-proteasome pathway using disulfiram and RNA interference and investigated the mechanisms underlying disulfiram in ccRCC. According to data from The Cancer Genome Atlas, NPL4 mRNA expression was significantly upregulated in clinical ccRCC samples compared with that in normal kidney samples, and patients with high NPL4 expression had poor overall survival compared with patients with low NPL4 expression. Disulfiram and NPL4 siRNA inhibited ccRCC cell proliferation in vitro, and disulfiram inhibited ccRCC tumor growth in a xenograft model. Synergistic antiproliferative effects were observed for combination treatment with disulfiram and sunitinib in vitro and in vivo. In RCC cells from mice treated with disulfiram and/or sunitinib, several genes associated with serine biosynthesis and aldose reductase were downregulated in cells treated with disulfiram or sunitinib alone and further downregulated in cells treated with both disulfiram and sunitinib. These findings provided insights into the mechanisms of disulfiram and suggested novel therapeutic strategies for RCC treatment.
Based on the microRNA (miRNA) signature of bladder cancer (BC) by deep sequencing, we recently found that several double-stranded mature miRNAs derived from the same pre-miRNAs were sufficiently ...expressed and acted as tumour suppressors by regulating common target genes in BC. Our deep-sequencing signature of BC showed that all miR-199 family members (miR-199a-3p/-5p and miR-199b-3p/-5p) were also downregulated. We hypothesised that these miRNAs may function as tumour suppressors by regulating common target genes.
Functional assays of BC cells were performed using transfection of mature miRNA. In silico analyses and luciferase reporter analyses were applied to identify target genes of these miRNAs. The overall survival of patients with BC in The Cancer Genome Atlas (TCGA) database was evaluated by the Kaplan-Meier method.
Restoration of these miRNAs significantly inhibited cell migration and invasion in BC cells. Integrin α3 (ITGA3) was directly regulated by these miRNAs. The Cancer Genome Atlas database showed that patients with low pre-miR-199 family (miR-199a-1/-2 and miR-199b) expression exhibited significantly poorer overall survival compared with patients with high pre-miR-199 family expression.
miR-199 family miRNAs functioned as tumour suppressors in BC cells by targeting ITGA3 and might be good prognostic markers for predicting survival in patients with BC.
Exosomes are 40-100 nm nano-sized extracellular vesicles. They are released from many cell types and move into the extracellular space, thereby transferring their components to recipient cells. ...Exosomes are receiving increasing attention as novel structures participating in intracellular communication. RAB27B is one of the leading proteins involved in exosome secretion, and oncogenic effects have been reported in several cancers. In recent years, molecularly targeted agents typified by sunitinib are widely used for the treatment of metastatic or recurrent renal cell carcinoma (RCC). However, intrinsic or acquired resistance to sunitinib has become a major issue. The present study aimed to elucidate the role of RAB27B in RCC including sunitinib-resistant and its role in exosomes. Bioinformatic analyses revealed that high expression of RAB27B correlates with progression of RCC. The expression of RAB27B protein in RCC cell lines was significantly enhanced compared with that in normal kidney cell lines. Furthermore, RAB27B protein expression was enhanced in all of the tested sunitinib-resistant RCC cell lines compared to parental cells. Although no specific effect of RAB27B on exosomes was identified in RCC cells, loss-of-function studies demonstrated that knockdown of RAB27B suppressed cell proliferation, migration and invasive activities. Moreover, anti-tumor effects of RAB27B downregulation were also observed in sunitinib-resistant RCC cells. RNA sequence and pathway analysis suggested that the oncogenic effects of RAB27B might be associated with MAPK and VEGF signaling pathways. These results showed that RAB27B is a prognostic marker and a novel therapeutic target in sunitinib-sensitive and -resistant RCCs. Further analyses should improve our understanding of sunitinib resistance in RCC.
Morphological changes in neuromuscular junctions (NMJs), which are synapses formed between α-motor neurons and skeletal muscle fibers, are considered to be important in age-related motor dysfunction. ...We have previously shown that the intake of dietary milk fat globule membrane (MFGM) combined with exercise attenuates age-related NMJ alterations in the early phase of aging. However, it is unclear whether the effect of MFGM with exercise on age-related NMJ alterations persists into old age, and whether intervention from old age is still effective when age-related changes in NMJs have already occurred. In this study, 6- or 18-month-old mice were treated with a 1% MFGM diet and daily running wheel exercise until 23 or 24 months of age, respectively. MFGM treatment with exercise was effective in suppressing the progression of age-related NMJ alterations in old age, and even after age-related changes in NMJs had already occurred. Moreover, the effect of MFGM intake with exercise was not restricted to NMJs but extended to the structure and function of peripheral nerves. This study demonstrates that MFGM intake with exercise may be a novel approach for improving motor function in the elderly by suppressing age-related NMJ alterations.
The active form of the small GTPase RAS binds to downstream effectors to promote cell growth and proliferation. RAS signal enhancement contributes to tumorigenesis, invasion, and metastasis in ...various different cancers. HRAS proto-oncogene GTPase (HRAS), one of the RAS isoforms, was the first human oncogene for which mutations were reported in T24 bladder cancer (BC) cells in 1982, and HRAS mutation or upregulation has been reported in several cancers. According to data from The Cancer Genome Atlas, HRAS expression was significantly upregulated in clinical BC samples compared to healthy samples (P=0.0024). HRAS expression was also significantly upregulated in BC with HRAS mutation compared to patients without HRAS mutation (P<0.0001). The tumor suppressive effect of salirasib, a RAS inhibitor, has been reported in several cancer types, but only at relatively high concentrations. As such, RAS inhibitors have not been used for clinical applications. The aim of the current study was to investigate the therapeutic potential of targeting HRAS using salirasib and small interfering RNA (siRNA) and to characterize the mechanism by which HRAS functions using recently developed quantitative in vitro proteome-assisted multiple reaction monitoring for protein absolute quantification (iMPAQT), in BC cells. iMPAQT allows measurement of the absolute abundance of any human protein with the high quantitative accuracy. Salirasib and siRNA targeting of HRAS inhibited cell proliferation, migration and invasion in HRAS wild type and HRAS-mutated cell lines. Proteomic analyses revealed that several metabolic pathways, including the oxidative phosphorylation pathway and glycolysis, were significantly downregulated in salirasib-treated BC cells. However, the expression levels of hexokinase 2, phosphoglycerate kinase 1, pyruvate kinase, muscle (PKM)l, PKM2 and lactate dehydrogenase A, which are downstream of RAS and target genes of hypoxia inducible factor-la, were not notably downregulated, which may explain the high concentration of salirasib required to inhibit cell viability. These findings provide insight into the mechanisms of salirasib, and suggest the need for novel therapeutic strategies to treat cancers such as BC. Key words: bladder cancer, HRAS proto-oncogene GTPase, RAS inhibitor, salirasib, in vitro proteome-assisted multiple reaction monitoring for protein absolute quantification
d‐3‐Phosphoglycerate dehydrogenase (PHGDH) conducts an important step in the synthesis of serine. Importantly, the PHGDH gene is often amplified in certain cancers. Our previous studies revealed that ...PHGDH gene amplification was associated with poor overall survival in clear cell renal cell carcinoma (ccRCC) and that metabolic reprogramming of serine synthesis through PHGDH recruitment allowed ccRCC cells to survive in unfavorable environments. There have been no investigations of the role of PHGDH expression in bladder cancer (BC). In this investigation, we examined the clinical importance of PHDGH in BC. Furthermore, we asked whether PHGDH expression could be exploited for BC therapy. Finally, we investigated the regulatory mechanisms that modulated the expression of PHGDH. Using data from The Cancer Genome Atlas, we found that patients with high‐grade BC had significantly higher PHGDH expression levels than did those with low‐grade BC. In addition, patients with high PHGDH expression did not survive as long as those with low expression. PHGDH downregulation by si‐RNAs or an inhibitor in BC cell lines significantly inhibited proliferative ability and induced apoptosis. Furthermore, combined treatment using a PHGDH inhibitor and gemcitabine/cisplatin achieved synergistic tumor suppression compared to use of a single agent both in vitro as well as in vivo. Mechanistic analyses of PHGDH regulation showed that PHGDH expression might be associated with DNA copy number and hypomethylation in BC. These findings suggest novel therapeutic strategies could be used in BC. Finally, our data enhance our understanding of the role of PHGDH in BC.
This research investigated PHGDH, a key enzyme in the serine biosynthesis, in bladder cancer. PHGDH downregulation significantly inhibited cell proliferation, and the combined treatment between PHGDH inhibitor and gemcitabine/cisplatin achieved synergistic tumor suppressive effect compared to each single agent group both in vitro and in vivo. We also indicated that PHGDH was regulated by DNA copy number and hypomethylation.
Motor units are generally recruited from the smallest to the largest following the size principle, while cutaneous stimulation has the potential to affect spinal motor control. We aimed to examine ...the effects of stimulating transient receptor potential channel sub-family M8 (TRPM8) combined with exercise on the modulation of spinal motor neuron (MN) excitability. Mice were topically administrated 1.5% icilin on the hindlimbs, followed by treadmill stepping. Spinal cord sections were immunostained with antibodies against c-fos and choline acetyltransferase. Icilin stimulation did not change the number of c-fos
MNs, but increased the average soma size of the c-fos
MNs during low-speed treadmill stepping. Furthermore, icilin stimulation combined with stepping increased c-fos
cholinergic interneurons near the central canal, which are thought to modulate MN excitability. These findings suggest that TRPM8-mediated cutaneous stimulation with low-load exercise promotes preferential recruitment of large MNs and is potentially useful as a new training method for rehabilitation.
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