DLX4

Gene Summary

Gene:DLX4; distal-less homeobox 4
Aliases: BP1, DLX7, DLX8, DLX9, OFC15
Location:17q21.33
Summary:Many vertebrate homeo box-containing genes have been identified on the basis of their sequence similarity with Drosophila developmental genes. Members of the Dlx gene family contain a homeobox that is related to that of Distal-less (Dll), a gene expressed in the head and limbs of the developing fruit fly. The Distal-less (Dlx) family of genes comprises at least 6 different members, DLX1-DLX6. The DLX proteins are postulated to play a role in forebrain and craniofacial development. Three transcript variants have been described for this gene, however, the full length nature of one variant has not been described. Studies of the two splice variants revealed that one encoded isoform functions as a repressor of the beta-globin gene while the other isoform lacks that function. [provided by RefSeq, Jul 2008]
Databases:OMIM, HGNC, Ensembl, GeneCard, Gene
Protein:homeobox protein DLX-4
Source:NCBIAccessed: 30 August, 2019

Ontology:

What does this gene/protein do?
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Cancer Overview

Research Indicators

Publications Per Year (1994-2019)
Graph generated 31 August 2019 using data from PubMed using criteria.

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

Tag cloud generated 30 August, 2019 using data from PubMed, MeSH and CancerIndex

Specific Cancers (6)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: DLX4 (cancer-related)

Zhang L, Wan Y, Jiang Y, et al.
Overexpression of BP1, an isoform of Homeobox Gene DLX4, promotes cell proliferation, migration and predicts poor prognosis in endometrial cancer.
Gene. 2019; 707:216-223 [PubMed] Related Publications
The expression of homeobox gene DLX4 has been verified in some tumors, but not in endometrial cancer. We found that expression of DLX7, a splicing isoform of DLX4, did not show any significant difference in expression between endometrial cancer and endometrium. However, BP1, another splicing isoform of DLX4, was highly expressed in endometrial cancer, and its expression was positively correlated with patient prognosis, cancer pathological grade, tumor invasion and metastasis. Lentiviral-mediated expression of BP1 in HEC-1-B cells accelerated the cell cycle progression from G0/G1 into S phase, and promoted cell proliferation and migration both in vitro and in vivo. Real-time PCR and western blotting showed that the expression levels of p15, p21 and E-cadherin significantly decreased, and levels of cyclinD1 and MMP-2 increased in endometrial cancer cells. In conclusion, our results demonstrate that high expression of BP1 is associated with poor prognosis in patients with endometrial cancer and promotes cell proliferation and migration.

Rivera-Calderón LG, Fonseca-Alves CE, Kobayashi PE, et al.
p-mTOR, p-4EBP-1 and eIF4E expression in canine prostatic carcinoma.
Res Vet Sci. 2019; 122:86-92 [PubMed] Related Publications
The mTOR/4E-BP1/eIF4E pathway plays important roles in the neoplastic transformation process and in tumour growth. In men, the mTOR/4E-BP1/eIF4E pathway was described as altered in different tumours, including prostate cancer (PC). Apart from humans, the dog is the only species that develops PC with high frequency and is considered a good model for comparative oncology initiatives. Due to limited information on this pathway in canine tumours, this study aimed to investigate mTOR, 4E-BP1 and eIF4E gene and protein expression in canine PC, as well as in metastatic and normal prostatic tissues, and to evaluate the correlations between gene/protein expression and Gleason score (GS) in PC. A total of 35 formalin-fixed paraffin-embedded (FFPE) samples, including 13 of normal prostatic tissue, 17 PC samples and 5 metastasis samples, were evaluated by immunohistochemistry and qPCR. mTOR gene mutation in the kinase domain was also investigated. We identified higher p-mTOR and eIF4E protein levels in canine PC with higher GS values (≥ 8) and a significant positive correlation in expression between these proteins. eIF4E overexpression was observed in metastasis relative to expression in normal samples. Our data suggest that p-mTOR and eIF4E expression is positively correlated with GS in canine PC, similar to the pattern in humans. More studies of the mTOR/4EBP1/eIF4E pathway should be performed to identify possible correlations of the proteins involved with clinical and pathologic findings in canine PC and the roles of these proteins as therapeutic targets for the treatment of canine PC.

Cheng C, Xiaohua W, Ning J, et al.
MiR-122 exerts anti-proliferative and apoptotic effects on nasopharyngeal carcinoma cells via the PI3K/AKT signaling pathway.
Cell Mol Biol (Noisy-le-grand). 2018; 64(13):21-25 [PubMed] Related Publications
To investigate the effects of microRNA-122 (miR-122) on the proliferation and apoptosis of nasopharyngeal carcinoma (NPC) HONE-1 cells, and its correlation with the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway. Human NPC cell line (HONE-1) was transfected with miR-122 inhibitor (anti-miR-122 group), negative controls (vector control group) via lipofectamines, and HONE-1 cell lines undergoing no transfection were selected (non-transfection group). The expression of miR-122, cell proliferation, apoptosis, and expressions of PI3K/AKT pathway and downstream target proteins in the three groups were determined using fluorescence quantitative polymerase chain reaction (qPCR), cell counting kit-8 (CCK8), immunofluorescence (IF) and Western blotting, respectively. The expression of miR-122 in the anti-miR-122 group was significantly lower than corresponding expressions in the non-transfection and vector control groups after 48h of transfection (p <0.05). The proliferation of cells in the anti-miR-122 group was significantly reduced with time after transfection (p <0.05). After 48h of transfection, the extent of apoptosis in the anti-miR-122 group (47.11 ± 1.95%) was significantly higher than that in normal control (7.37 ± 0.82%) and vector control group (8.54 ± 0.96%; p <0.05). There were no significant differences in the expressions of PI3K, AKT, mTOR protein, and the downstream signal proteins (p70S6K and 4E-BP1) in the three groups (p >0.05). However, the expressions of phosphorylated forms of these proteins were significantly lower in the anti-miR-122 group than in the non-transfection and vector control groups (p <0.05). IF results revealed that there were no significant differences in the fluorescence intensity value of PI3K and Akt among the three groups of patients (p>0.05). Inhibition of the expression of miR-122 in NPC suppresses the proliferation, and promotes their apoptosis through the PI3K/AKT signal transduction pathway.

Hsu FF, Chou YT, Chiang MT, et al.
Signal peptide peptidase promotes tumor progression via facilitating FKBP8 degradation.
Oncogene. 2019; 38(10):1688-1701 [PubMed] Related Publications
Signal peptide peptidase (SPP) is an endoplasmic reticulum (ER)-resident aspartyl protease mediating intramembrane cleavage of type II transmembrane proteins. Increasing evidence has supported the role of SPP in ER-associated protein degradation. In the present study, we show that SPP expression is highly induced in human lung and breast cancers and correlated with disease outcome. Stable depletion of SPP expression in lung and breast cancer cell lines significantly reduced cell growth and migration/invasion abilities. Quantitative analysis of the proteomic changes of microsomal proteins in lung cancer cells by the stable isotope labeling with amino acids in cell culture (SILAC) approach revealed that the level of FKBP8, an endogenous inhibitor of mTOR, was significantly increased following SPP depletion. Co-immunoprecipitation assay and confocal immunofluorescence demonstrated that SPP interacted and colocalized with FKBP8 in ER, supporting that FKBP8 is a protein substrate of SPP. Cycloheximide chase and proteasome inhibition experiments revealed that SPP-mediated proteolysis facilitated FKBP8 protein degradation in cytosol. Further experiment demonstrated that the levels of phosphorylation in mTOR and its downstream effectors, S6K and 4E-BP1, were significantly lower in SPP-depleted cells. The reduced mTOR signaling and decreases of growth and migration/invasion abilities induced by SPP depletion in cancer cells could be reversed by FKBP8 downregulation. The implication of FKBP8 in SPP-mediated tumorigenicity was also observed in the xenograft model. Together, these findings disclose that SPP promotes tumor progression, at least in part, via facilitating the degradation of FKBP8 to enhance mTOR signaling.

Hu M, Liu L, Yao W
Activation of p53 by costunolide blocks glutaminolysis and inhibits proliferation in human colorectal cancer cells.
Gene. 2018; 678:261-269 [PubMed] Related Publications
Colorectal cancer is a leading cause of cancer-related death. Glutaminolysis has been suggested as a therapeutic target for cancer. Costunolide is a natural sesquiterpene lactone showing potent antitumor activity. Our studies were aimed at evaluating how costunolide affected glutaminolysis leading to proliferation inhibition in human colorectal cancer cells. Costunolide suppressed viability and proliferation of HCT116 cells concentration-dependently, but did not apparently affect human intestinal epithelial cells. Costunolide at 20 μM reduced viability and proliferation of HCT116 cells time-dependently. Costunolide also repressed phosphorylation of mTOR and its downstream kinases p70S6K and 4E-BP1. Examinations of glutaminolysis metabolites showed that costunolide increased intracellular glutamine levels, but decreased intracellular levels of glutamate, α-ketoglutarate (α-KG), and ATP in HCT116 cells, suggesting costunolide blockade of glutaminolysis. Furthermore, costunolide inhibited promoter activity of glutaminase 1 (GLS1), the first rate-limiting enzyme in glutaminolysis, and reduced mRNA and protein expression of GLS1 in HCT116 cells, The GLS1 inhibitor BPTES, similar to costunolide, significantly reduced intracellular levels of α-KG and ATP and inhibited proliferation in HCT116 cells. Finally, costunolide increased phosphorylation and nuclear translocation of p53 in HCT116 cells. Both p53 inhibitor pifithrin-α and p53 siRNA significantly rescued costunolide suppression of GLS1 promoter activity and expression in HCT116 cells. These data in aggregate suggested that activation of p53 was required for costunolide inhibition of GLS1 resulting in blockade of glutaminolysis and inhibition of proliferation in colorectal cancer cells, which was a novel mechanism underlying the antitumor activity of costunolide against colorectal cancer.

Suk FM, Chang CC, Lin RJ, et al.
Int J Mol Sci. 2018; 19(5) [PubMed] Free Access to Full Article Related Publications
Monocyte chemotactic protein induced protein 3 (MCPIP3) belongs to the Cys⁻Cys⁻Cys⁻His (CCCH)-zinc finger protein family and contains a highly conserved CCCH-zinc finger domain and a Nedd4-BP1 YacP nuclease (NYN) domain. Previous studies showed that MCPIP3 inhibits the expression of proinflammatory genes, such as vascular cell adhesion molecule (

Lou Y, Fallah Y, Yamane K, Berg PE
BP1, a potential biomarker for breast cancer prognosis.
Biomark Med. 2018; 12(5):535-545 [PubMed] Related Publications
Homeobox genes are critical in tumor development. An isoform protein of DLX4 called BP1 is expressed in 80% of invasive ductal breast carcinomas. BP1 overexpression is implicated in an aggressive phenotype and poor prognosis. BP1 upregulation is associated with estrogen receptor negativity so those tumors do not respond to antiestrogens. Breast cancer is the second leading cause of death in women. BP1 could serve as both a novel prognostic biomarker for breast cancer and a therapeutic target. In this review, we address the role of BP1 protein in tumorigenesis of breast cancer and four other malignancies. A number of functions of BP1 in cancer are also discussed.

Ding M, Van der Kwast TH, Vellanki RN, et al.
The mTOR Targets 4E-BP1/2 Restrain Tumor Growth and Promote Hypoxia Tolerance in PTEN-driven Prostate Cancer.
Mol Cancer Res. 2018; 16(4):682-695 [PubMed] Related Publications
The mTOR signaling pathway is a central regulator of protein synthesis and cellular metabolism in response to the availability of energy, nutrients, oxygen, and growth factors. mTOR activation leads to phosphorylation of multiple downstream targets including the eukaryotic initiation factor 4E (eIF4E) binding proteins-1 and -2 (EIF4EBP1/4E-BP1 and EIF4EBP2/4E-BP2). These binding proteins inhibit protein synthesis, but are inactivated by mTOR to stimulate cell growth and metabolism. However, the role of these proteins in the context of aberrant activation of mTOR, which occurs frequently in cancers through loss of PTEN or mutational activation of the PI3K/AKT pathway, is unclear. Here, even under conditions of aberrant mTOR activation, hypoxia causes dephosphorylation of 4E-BP1/4E-BP2 and increases their association with eIF4E to suppress translation. This is essential for hypoxia tolerance as knockdown of 4E-BP1 and 4E-BP2 decreases proliferation under hypoxia and increases hypoxia-induced cell death. In addition, genetic deletion of 4E-BP1 and 4E-BP2 significantly accelerates all phases of cancer development in the context of PTEN loss-driven prostate cancer in mice despite potent PI3K/AKT and mTOR activation. However, even with a more rapid onset, tumors that establish in the absence of 4E-BP1 and 4E-BP2 have reduced levels of tumor hypoxia and show increased cell death within hypoxic tumor regions. Together, these data demonstrate that 4E-BP1 and 4E-BP2 act as essential metabolic breaks even in the context of aberrant mTOR activation and that they are essential for the creation of hypoxia-tolerant cells in prostate cancer.

Huang Z, Fang W, Liu W, et al.
Aspirin induces Beclin-1-dependent autophagy of human hepatocellular carcinoma cell.
Eur J Pharmacol. 2018; 823:58-64 [PubMed] Related Publications
Aspirin not only reduces the incidence of hepatocellular carcinoma (HCC) but also plays a synergistic role with chemotherapy for HCC treatment. However, the underlying mechanisms remain incompletely elucidated. Given that autophagy triggers cancer cell death, the present study examined the autophagic effect of aspirin on HCC cells. Results showed that aspirin increased LC3II/LC3I ratio, decreased p62 expression, and enhanced autophagic flux (autophagosome and autolysosome puncta) in Hep3B, HepG2, or SMMC-7721 cells, reflecting the autophagy of HCC cells. The autophagic effects of aspirin depended on Beclin-1 expression. Aspirin disrupted the interaction between Bcl-2 and Beclin-1. In addition to activating the AMP-activated protein kinase, c-Jun N-terminal kinase, and Glycogen synthase kinase-3 pathways, aspirin inhibited the mammalian-target-of rapamycin-S6K1/4E-BP1 signaling. Aspirin induced autophagy of HCC cell. This study contributes to understanding the chemoprotective and inhibitory effects of aspirin on HCC development.

Wang J, Ye Q, Cao Y, et al.
Snail determines the therapeutic response to mTOR kinase inhibitors by transcriptional repression of 4E-BP1.
Nat Commun. 2017; 8(1):2207 [PubMed] Free Access to Full Article Related Publications
Loss of 4E-BP1 expression has been linked to cancer progression and resistance to mTOR inhibitors, but the mechanism underlying 4E-BP1 downregulation in tumors remains unclear. Here we identify Snail as a strong transcriptional repressor of 4E-BP1. We find that 4E-BP1 expression inversely correlates with Snail level in cancer cell lines and clinical specimens. Snail binds to three E-boxes present in the human 4E-BP1 promoter to repress transcription of 4E-BP1. Ectopic expression of Snail in cancer cell lines lacking Snail profoundly represses 4E-BP1 expression, promotes cap-dependent translation in polysomes, and reduces the anti-proliferative effect of mTOR kinase inhibitors. Conversely, genetic and pharmacological inhibition of Snail function restores 4E-BP1 expression and sensitizes cancer cells to mTOR kinase inhibitors by enhancing 4E-BP1-mediated translation-repressive effect on cell proliferation and tumor growth. Our study reveals a critical Snail-4E-BP1 signaling axis in tumorigenesis, and provides a rationale for targeting Snail to improve mTOR-targeted therapies.

Ma M, Dai J, Xu T, et al.
Analysis of TSC1 mutation spectrum in mucosal melanoma.
J Cancer Res Clin Oncol. 2018; 144(2):257-267 [PubMed] Related Publications
PURPOSE: Mucosal melanoma is a relatively rare subtype of melanoma for which no clearly established therapeutic strategy exists. The genes of the mTOR signalling pathway have drawn great attention as key targets for cancer treatment, including melanoma. In this study, we aimed to investigate the mutation status of the upstream mTOR regulator TSC1 and evaluated its correlation with the clinicopathological features of mucosal melanoma.
METHODS: We collected 91 mucosal melanoma samples for detecting TSC1 mutations. All the coding exons of TSC1 were amplified by PCR and subjected to Sanger sequencing. Expression level of TSC1 encoding protein (hamartin) was detected by immunohistochemistry. The activation of mTOR pathway was determined by evaluating the phosphorylation status of S6RP and 4E-BP1.
RESULTS: The overall mutation frequency of TSC1 was found to be 17.6% (16/91 patients). TSC1 mutations were more inclined to occur in advanced mucosal melanoma (stages III and IV). In the 16 patients with TSC1 mutations, 14 different mutations were detected, affecting 11 different exons. TSC1 mutations were correlated with upregulation of S6RP phosphorylation but were unrelated to 4E-BP1 phosphorylation or hamartin expression. Mucosal melanoma patients with TSC1 mutations had a worse outcome than patients without TSC1 mutations (24.0 versus 34.0 months, P = 0.007).
CONCLUSIONS: Our findings suggest that TSC1 mutations are frequent in mucosal melanoma. TSC1 mutations can activate the mTOR pathway through phospho-S6RP and might be a poor prognostic predictor of mucosal melanoma. Our data implicate the potential significance of TSC1 mutations for effective and specific drug therapy for mucosal melanoma.

Zhao Y, Wang Y, Wang Y
Up-regulated
Biosci Rep. 2017; 37(6) [PubMed] Free Access to Full Article Related Publications
It has been shown that

De A, Jacobson BA, Peterson MS, et al.
4EGI-1 represses cap-dependent translation and regulates genome-wide translation in malignant pleural mesothelioma.
Invest New Drugs. 2018; 36(2):217-229 [PubMed] Related Publications
Deregulation of cap-dependent translation has been implicated in the malignant transformation of numerous human tissues. 4EGI-1, a novel small-molecule inhibitor of cap-dependent translation, disrupts formation of the eukaryotic initiation factor 4F (eIF4F) complex. The effects of 4EGI-1-mediated inhibition of translation initiation in malignant pleural mesothelioma (MPM) were examined. 4EGI-1 preferentially inhibited cell viability and induced apoptosis in MPM cells compared to normal mesothelial (LP9) cells. This effect was associated with hypophosphorylation of 4E-binding protein 1 (4E-BP1) and decreased protein levels of the cancer-related genes, c-myc and osteopontin. 4EGI-1 showed enhanced cytotoxicity in combination with pemetrexed or gemcitabine. Translatome-wide polysome microarray analysis revealed a large cohort of genes that were translationally regulated upon treatment with 4EGI-1. The 4EGI-1-regulated translatome was negatively correlated to a previously published translatome regulated by eIF4E overexpression in human mammary epithelial cells, which is in agreement with the notion that 4EGI-1 inhibits the eIF4F complex. These data indicate that inhibition of the eIF4F complex by 4EGI-1 or similar translation inhibitors could be a strategy for treating mesothelioma. Genome wide translational profiling identified a large cohort of promising target genes that should be further evaluated for their potential significance in the treatment of MPM.

Jin L, Jin MH, Nam AR, et al.
Anti-tumor effects of NVP-BKM120 alone or in combination with MEK162 in biliary tract cancer.
Cancer Lett. 2017; 411:162-170 [PubMed] Related Publications
There are currently no clinically validated therapeutic targets for biliary tract cancer (BTC). Despite promising results in other cancers, compounds targeting the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, alone or in combination with Ras/Raf/MEK pathway inhibitors, have not been evaluated in BTC. Here, we examined the effects of a pan-PI3K inhibitor (BKM120) with or without a MEK inhibitor (MEK162), on eight human BTC cell lines carrying mutations in K-Ras and/or the PI3K catalytic subunit, PI3KCA. BKM120 inhibited the colony-forming ability and migration of BTC cells carrying wild-type (WT) PI3KCA and either mutant (MT) or WT K-Ras, but not of cells carrying mutations in both genes. In K-Ras-WT cells, BKM120 decreased the phosphorylation of Akt, its downstream effector kinase p70S6K, and the translational repressor 4E-BP1. Interestingly, BKM120 did not induce cell cycle arrest or suppress PI3K signaling via restoration of p-4E-BP1 in cells with PIK3CA and K-Ras double mutations. Notably, the resistance of dual K-Ras/PI3KCA-mutant cells to BKM120 was overcome by treatment with a combination of BKM120 and MEK162. Our findings thus support the clinical development of BKM120 monotherapy or BKM120/MEK162 combination therapy for the treatment of BTC.

Wang S, Li J, Du Y, et al.
The Class I PI3K inhibitor S14161 induces autophagy in malignant blood cells by modulating the Beclin 1/Vps34 complex.
J Pharmacol Sci. 2017; 134(4):197-202 [PubMed] Related Publications
S14161 is a pan-Class I PI3K inhibitor that induces blood cancer cell death, but its mechanism is largely unknown. In the present study, we evaluated the role of S14161 in autophagy, an emerging event in cell destination. Multiple myeloma cell lines RPMI-8226, OPM2, KMS11 and leukemia cell line K562 were treated with S14161. The results showed that S14161 induced autophagy as demonstrated by increased LC3-II and decreased p62, which were prevented by autophagy inhibitors including 3-methyladenine and bafilomycin A1. Mechanistic studies showed that S14161 had no effects on Vps34 expression, but increased Beclin 1 and decreased Bcl-2, two major regulators of autophagy. Furthermore, S14161 dissociated the Beclin 1/Bcl-2 complex and enhanced the formation of Beclin 1/Vps34 complex. Moreover, S14161 inhibited the mTORC1 signaling transduction. S14161 downregulated activation of mTOR and its two critical targets 4E-BP1 and p70S6K, suggesting S14161 inhibited protein synthesis. Taken together, these results demonstrated that Class I PI3K regulates autophagy by modulating protein synthesis and the Beclin 1 signaling pathway. This finding helps understanding the roles of PI3K in autophagy and cancer treatment.

Feng J, Qi B, Guo L, et al.
miR-382 functions as a tumor suppressor against esophageal squamous cell carcinoma.
World J Gastroenterol. 2017; 23(23):4243-4251 [PubMed] Free Access to Full Article Related Publications
AIM: To explore the effect of miR-382 on esophageal squamous cell carcinoma (ESCC)
METHODS: Eca109 cells derived from human ESCC and Het-1A cells derived from human normal esophageal epithelium were used. Lentivirus-mediated miR-382 was overexpressed in Eca109 cells. The effect of miR-382 on cell proliferation was evaluated by MTT and colony formation assay. For cell cycle analysis, cells were fixed and stained for 30 min with propidium iodide (PI) staining buffer containing 10 mg/mL PI and 100 mg/mL RNase A, and analyzed by BD FACSCalibur™ flow cytometer. For cell apoptosis assay, cells were stained with an Annexin V-FITC/PI Apoptosis Detection Kit according to the manufacturer's instructions and analyzed by a dual-laser flow cytometer. Cell invasion and migration abilities were determined through use of transwell chambers, non-coated or pre-coated with matrigel. Levels of proteins related to cell growth and migration were examined by western blotting.
RESULTS: Endogenous miR-382 was down-regulated in Eca109 cells compared with Het-1A. Introduction of miR-382 not only significantly inhibited proliferation and colony formation, but also arrested cell cycle at the G2/M phase, as well as promoted apoptosis and autophagy in Eca109 cells. Migration, invasion and epithelial-mesenchymal transition of Eca109 cells were suppressed by overexpressing miR-382. Western blotting results showed that miR-382 inhibited the phosphorylation of mTOR and 4E-BP1.
CONCLUSION: miR-382 functions as a tumor suppressor against ESCC development and metastasis, and could be considered as a potential drug source for the treatment of ESCC patients.

Li MY, Mi C, Wang KS, et al.
Shikonin suppresses proliferation and induces cell cycle arrest through the inhibition of hypoxia-inducible factor-1α signaling.
Chem Biol Interact. 2017; 274:58-67 [PubMed] Related Publications
Hypoxia enhances the development of solid tumors. Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that is dominantly expressed under hypoxia in solid tumor cells and is a key factor of tumor regulation. HIF-1α regulates several target genes involved in many aspects of cancer progression, including angiogenesis, metastasis, and cell proliferation, as well as imparting resistance to cancer treatment. In this study, we assessed shikonin, which derives from the traditional medical herb Lithospermum erythrorhizon, for its anti-cancer effects in hypoxia-induced human colon cancer cell lines. Shikonin showed potent inhibitory activity against hypoxia-induced HIF-1α activation in various human cancer cell lines and efficient scavenging activity of hypoxia-induced reactive oxygen species in tumor cells. Further analysis revealed that shikonin inhibited HIF-1α protein synthesis without affecting the expression of HIF-1α mRNA or degrading HIF-1α protein. It was subsequently shown to attenuate the activation of downstream mTOR/p70S6K/4E-BP1/eIF4E kinase. Shikonin also dose-dependently caused the cell cycle arrest of activated HCT116 cells and inhibited the proliferation of HCT116 and SW620 cells. Moreover, it significantly inhibited tumor growth in a xenograft modal. These findings suggest that shikonin could be considered for use as a potential drug in human colon cancer therapy.

Chuang CK, Lin HCA, Tasi HY, et al.
Clinical presentations and molecular studies of invasive renal epithelioid angiomyolipoma.
Int Urol Nephrol. 2017; 49(9):1527-1536 [PubMed] Related Publications
PURPOSE: Epithelioid angiomyolipoma (EAML) is a rare variant of renal angiomyolipoma with malignant potential, and the cytogenetic and clinical behavior of EAML remains a challenging issue.
METHODS: We retrospectively analyze the clinical courses of five EAML, the use of everolimus on metastatic EAML, and next-generation sequencing (NGS) and polymerase chain reaction (PCR) studies to investigate the gene mutation of TSC and the impact of PI3K/Akt/mTOR signaling pathway in metastatic EAML.
RESULTS: The mean age was 37.8 years, mean tumor size was 13 cm, all patients received radical nephrectomy, one stage IV patient received neoadjuvant mTOR inhibitor management, and one patient with high mitotic activity developed metastasis 1 year after nephrectomy. NGS assay showed a frameshift gene mutation of TSC2 in chromosome 16. PCR array for the mRNA alterations in PI3K/Akt/mTOR signaling pathway of EAML showed high expression of PIP3, AKT, TSC1, mTOR, PDK1, P70, 4E-BP1 and elF4E.
CONCLUSION: EAML of the kidney is a specific type of renal AML with malignant potentials, where around 22% of the patients present with invasion or metastasis. Higher mitotic activities indicate a greater metastatic potential, with radical nephrectomy as the treatment of choice, and mTOR inhibitors such as everolimus either as neoadjuvant or adjuvant targeted therapy can lead to a better clinical outcome. NGS to explore the mTOR signaling pathway may help us to better understand the pathogenesis and progression of EAML.

Persaud L, Zhong X, Alvarado G, et al.
eIF2α Phosphorylation Mediates IL24-Induced Apoptosis through Inhibition of Translation.
Mol Cancer Res. 2017; 15(8):1117-1124 [PubMed] Free Access to Full Article Related Publications
IL24 is an immunomodulatory cytokine that also displays broad cancer-specific suppressor effects. The tumor-suppressor activities of IL24 include inhibition of angiogenesis, sensitization to chemotherapy, and cancer-specific apoptosis. Supra-physiologic activation and/or overexpression of translation initiation factors are implicated in the initiation and progression of cancer animal models as well as a subset of human cancers. Activation and/or overexpression of translation initiation factors correlate with aggressiveness of cancer and poor prognosis. Two rate-limiting translation initiation complexes, the ternary complex and the eIF4F complex, are regulated by eIF2α and 4E-BP1 phosphorylation, respectively. The work reported here provides direct evidence that IL24 induces inhibition of translation initiation leading to apoptosis in squamous cell carcinoma. A dominant constitutively active mutant of eIF2α, which is resistant to phosphorylation, was used to determine the involvement of eIF2α in IL24-induced apoptosis. Treatment with IL24 resulted in inhibition of protein synthesis, expression of downstream biomarkers of ternary complex depletion such as CHOP, and induction of apoptosis in cancer cells. The constitutively active nonphosphorylatable mutant of eIF2α, eIF2α-S51A, reversed both the IL24-mediated translational block and IL24-induced apoptosis. Intriguingly, IL24 treatment also caused hypophosphorylation of 4E-BP1, which binds to eIF4E with high affinity, thus preventing its association with eIF4G and therefore preventing elF4F complex assembly.

Li CF, Fang FM, Chen YY, et al.
Overexpressed Fatty Acid Synthase in Gastrointestinal Stromal Tumors: Targeting a Progression-Associated Metabolic Driver Enhances the Antitumor Effect of Imatinib.
Clin Cancer Res. 2017; 23(16):4908-4918 [PubMed] Related Publications

Hau AM, Nakasaki M, Nakashima K, et al.
Differential mTOR pathway profiles in bladder cancer cell line subtypes to predict sensitivity to mTOR inhibition.
Urol Oncol. 2017; 35(10):593-599 [PubMed] Related Publications
BACKGROUND: Molecular classification of bladder cancer has been increasingly proposed as a potential tool to predict clinical outcomes and responses to chemotherapy. Here we focused on mechanistic target of rapamycin (mTOR) inhibition as a chemotherapeutic strategy and characterized the expression profile of mTOR signaling targets in representative bladder cancer cell lines from basal, luminal, and either basal/luminal ("non-type") molecular subtypes.
MATERIALS AND METHODS: Protein and mRNA expression of mTOR signaling components from representative luminal (RT4 and RT112), basal (SCaBER and 5637), and nontype (T24 and J82) bladder cancer cell line subtypes were determined by Western blot and database mining analysis of the Cancer Cell Line Encyclopedia. Cell viability following treatment with either, Torin-2 or KU-0063794, 2 dual mTOR complex 1/2 inhibitors, was determined by MTT assay. Immunoblot analysis of cells treated with Torin-2 or KU-0063794 was performed to determine the effects of mTOR inhibition on expression and phosphorylation status of mTOR signaling components, Akt, 4E-BP1, and ribosomal protein S6.
RESULTS: Molecular subtypes of bladder cancer cell lines each exhibited a distinct pattern of expression of mTOR-associated genes and baseline phosphorylation level of Akt and 4E-BP1. Cells with low levels of Akt Ser-473 phosphorylation were more resistant to the cytotoxic effects of mTOR inhibition with Torin-2, but not KU-0063794. Exposure to Torin-2 and KU-0063794 both potently and rapidly inhibited phosphorylation of Akt Ser-473 and Thr-308, and 4E-BP1 T37/46 in cell lines that included basal and nontype subtypes.
CONCLUSIONS: Differential gene expression and protein activity associated with mTOR signaling is observed among bladder cancer cell lines stratified into basal, luminal, and nontype subtypes. Urothelial carcinomas characterized by high baseline Akt Ser-473 phosphorylation may be best suited for targeted mTOR therapies.

Batool A, Aashaq S, Andrabi KI
Reappraisal to the study of 4E-BP1 as an mTOR substrate - A normative critique.
Eur J Cell Biol. 2017; 96(4):325-336 [PubMed] Related Publications
mTOR-4E-BP1 axis is regarded as the best oncogenic circuitry impinging on translational control whereby mTORC1 dictates post-translational regulation of 4E-BP1. This review provides new insights into the molecular network of signalling pathways highlighting the recent explosion of studies in respect to the deviant behaviour of 4E-BP1 towards mTORC1. Despite the striking conservation of mTOR nexus, the eccentric phosphorylation dynamics of 4E-BP1 negate the apparent linear architecture of mTORC1 attesting the importance of other kinases that may evoke cross-talks with the conventional frame, most of which are enlisted in the manuscript. We also throw light on the tenuous role of rapamycin in 4E-BP1 regulation, which further necessitates the evaluation of 4E-BP1 to envisage the underlying molecular mechanisms in the discovery of novel drugs of 4E-BP1 for new treatment strategies. Finally, the review brings forward comprehensive studies delineating the redundancy of 4E-BP isoforms in regulating translational control.

Julius A, Desai A, Yung RL
Recombinant human erythropoietin stimulates melanoma tumor growth through activation of initiation factor eIF4E.
Oncotarget. 2017; 8(18):30317-30327 [PubMed] Free Access to Full Article Related Publications
Recombinant human erythropoietin (EPO) is standard treatment for anemia in cancer patients. Recent clinical trials suggest that EPO may accelerate tumor progression and increase mortality. However, the evidence supporting a growth-promoting effect of EPO has remained controversial. Employing an in vivo model of B16 murine melanoma, we observed that administration of EPO to tumor bearing C57BL/6 mice resulted in pronounced acceleration of melanoma growth. Our in vitro studies demonstrate that B16 murine melanoma cells express EPOR, both at the protein and mRNA levels. Interestingly, expression of EPOR was retained in the established tumors. EPO stimulation of B16 cells enhanced proliferation and protein synthesis rates, and correlated with activation of the receptor associated Janus kinase 2 (Jak2) as well as phosphorylation of extracellular signal-regulated kinase (Erk) 1/2 and Akt kinases. Treatment with EPO and Jak-2 antagonists significantly inhibited EPO-mediated B16 cell proliferation. Moreover, EPO dose-dependently induced the phosphorylation and activation of the translation initiation factor eIF4E as well as the phosphorylation of its repressor, the eIF4E binding protein 4E-BP1. Finally, using eIF4E small interfering RNA (siRNA), we observed that EPO-mediated stimulation of B16 cell proliferation is eIF4E-dependent. Our results indicate that EPO exerts a powerful stimulatory effect on cell proliferation and de novo protein synthesis in melanoma cells through activation of the initiation factor eIF4E.

Mi C, Ma J, Wang KS, et al.
Imperatorin suppresses proliferation and angiogenesis of human colon cancer cell by targeting HIF-1α via the mTOR/p70S6K/4E-BP1 and MAPK pathways.
J Ethnopharmacol. 2017; 203:27-38 [PubMed] Related Publications
ETHNOPHARMACOLOGICAL RELEVANCE: Angelica dahurica is a commonly used traditional Chinese medicine to treat migraine headache, toothache and cancer. Imperatorin is an active natural furocoumarin component originating from Angelica dahurica and has been shown to exhibit multiple bioeffector functions, including anti-cancer activity. However, the mechanism by which imperatorin inhibits tumor growth is not fully understood.
AIM OF THE STUDY: The aim of this study was to investigate the effectiveness of imperatorin as a treatment of cancer and to identify the underlying mechanisms of its anticancer activity.
MATERIALS AND METHODS: HCT116, HeLa, and Hep3B cells were used in this study. Major assays were promoter-reporter gene assay, MTT, western blot analysis, immunofluorescence assay, reverse transcription-PCR (RT-PCR), flow cytometric analysis, clonogenic assay, EdU labeling and immunofluorescence, xenografted assay, and VEGF ELISA.
RESULTS: We here demonstrated the effect of imperatorin on hypoxia-inducible factor-1 (HIF-1) activation. Imperatorin showed a potent inhibitory activity against HIF-1 activation induced by hypoxia in various human cancer cell lines. This compound markedly decreased the hypoxia-induced accumulation of HIF-1α protein dose-dependently, whereas it did not affect the expressions of HIF-1β and topoisomerase-I (Topo-I). Further analysis revealed that imperatorin inhibited HIF-1α protein synthesis, without affecting the expression level of HIF-1α mRNA or degradation of HIF-1α protein. Moreover, the phosphorylation levels of mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (p70S6K), eIF4E binding protein-1 (4E-BP1), eukaryotic initiation factor 4E (eIF4E), extracellular signal-regulated kinase-1/2 (ERK1/2), SAPK/JNK and p38 were significantly suppressed by imperatorin. Furthermore, imperatorin prevented hypoxia-induced expression of HIF-1 target genes and flow cytometric analysis indicated that imperatorin induced G1 phase arrest in human colon cancer cell (HCT116). We found that imperatorin administration inhibits tumor growth and blocks tumor angiogenesis in a xenograft tumor model.
CONCLUSIONS: These results show that imperatorin inhibited HIF-1α protein synthesis by downregulating the mTOR/p70S6K/4E-BP1 and MAPK pathways. These conclusions suggest that imperatorin is an effective inhibitor of HIF-1 and provide new perspectives into the mechanism of its anticancer activity.

Zeng Q, Qin S, Zhang H, et al.
Rapamycin attenuates BAFF-extended proliferation and survival via disruption of mTORC1/2 signaling in normal and neoplastic B-lymphoid cells.
J Cell Physiol. 2018; 233(1):516-529 [PubMed] Free Access to Full Article Related Publications
B cell activating factor from the TNF family (BAFF) stimulates B-cell proliferation and survival, but excessive BAFF promotes the development of aggressive B cells leading to malignant and autoimmune diseases. Recently, we have reported that rapamycin, a macrocyclic lactone, attenuates human soluble BAFF (hsBAFF)-stimulated B-cell proliferation/survival by suppressing mTOR-mediated PP2A-Erk1/2 signaling pathway. Here, we show that the inhibitory effect of rapamycin on hsBAFF-promoted B cell proliferation/survival is also related to blocking hsBAFF-stimulated phosphorylation of Akt, S6K1, and 4E-BP1, as well as expression of survivin in normal and B-lymphoid (Raji and Daudi) cells. It appeared that both mTORC1 and mTORC2 were involved in the inhibitory activity of rapamycin, as silencing raptor or rictor enhanced rapamycin's suppression of hsBAFF-induced survivin expression and proliferation/viability in B cells. Also, PP242, an mTORC1/2 kinase inhibitor, repressed survivin expression, and cell proliferation/viability more potently than rapamycin (mTORC1 inhibitor) in B cells in response to hsBAFF. Of interest, ectopic expression of constitutively active Akt (myr-Akt) or constitutively active S6K1 (S6K1-ca), or downregulation of 4E-BP1 conferred resistance to rapamycin's attenuation of hsBAFF-induced survivin expression and B-cell proliferation/viability, whereas overexpression of dominant negative Akt (dn-Akt) or constitutively hypophosphorylated 4E-BP1 (4EBP1-5A), or downregulation of S6K1, or co-treatment with Akt inhibitor potentiated the inhibitory effects of rapamycin. The findings indicate that rapamycin attenuates excessive hsBAFF-induced cell proliferation/survival via blocking mTORC1/2 signaling in normal and neoplastic B-lymphoid cells. Our data underscore that rapamycin may be a potential agent for preventing excessive BAFF-evoked aggressive B-cell malignancies and autoimmune diseases.

Liang G, Liu M, Wang Q, et al.
Itraconazole exerts its anti-melanoma effect by suppressing Hedgehog, Wnt, and PI3K/mTOR signaling pathways.
Oncotarget. 2017; 8(17):28510-28525 [PubMed] Free Access to Full Article Related Publications
Malignant melanoma is the deadliest form of all skin cancers. Itraconazole, a commonly used systemic antifungal drug, has been tested for its anti-tumor effects on basal cell carcinoma, prostate cancer, and non-small cell lung cancer. Whether itraconazole has any specific anti-tumor effect on melanoma remains unknown. However, the goal of this study is to investigate the effect of itraconazole on melanoma and to reveal some details of its underlying mechanism. In the in vivo xenograft mouse model, we find that itraconazole can inhibit melanoma growth and extend the survival of melanoma xenograft mice, compared to non-itraconazole-treated mice. Also, itraconazole can significantly inhibit cell proliferation, as demonstrated by Ki-67 staining in itraconazole-treated tumor tissues. In in vitro, we show that itraconazole inhibits the proliferation and colony formation of both SK-MEL-28 and A375 human melanoma cells. Moreover, we demonstrate that itraconazole significantly down-regulates Gli-1, Gli-2, Wnt3A, β-catenin and cyclin D1, while it up-regulates Gli-3 and Axin-1, indicating potent inhibitory effects of itraconazole on Hedgehog (Hh) and Wnt signaling pathways. Furthermore, itraconazole significantly suppresses the PI3K/mTOR signaling pathway - indicated by the down-regulated phosphorylation of p70S6K, 4E-BP1 and AKT - but has no effect on the phosphorylation of MEK or ERK. Our data suggest that itraconazole inhibits melanoma growth through an interacting regulatory network that includes Hh, Wnt, and PI3K/mTOR signaling pathways. These results suggest that this agent has several potent anti-melanoma features and may be useful in the synergesis of other anti-cancer drugs via blockage of the Hh, Wnt and PI3K/mTOR signaling pathways.

Jhanwar-Uniyal M, Amin AG, Cooper JB, et al.
Discrete signaling mechanisms of mTORC1 and mTORC2: Connected yet apart in cellular and molecular aspects.
Adv Biol Regul. 2017; 64:39-48 [PubMed] Related Publications
Activation of PI3K/Akt/mTOR (mechanistic target of rapamycin) signaling cascade has been shown in tumorigenesis of numerous malignancies including glioblastoma (GB). This signaling cascade is frequently upregulated due to loss of the tumor suppressor PTEN, a phosphatase that functions antagonistically to PI3K. mTOR regulates cell growth, motility, and metabolism by forming two multiprotein complexes, mTORC1 and mTORC2, which are composed of special binding partners. These complexes are sensitive to distinct stimuli. mTORC1 is sensitive to nutrients and mTORC2 is regulated via PI3K and growth factor signaling. mTORC1 regulates protein synthesis and cell growth through downstream molecules: 4E-BP1 (also called EIF4E-BP1) and S6K. Also, mTORC2 is responsive to growth factor signaling by phosphorylating the C-terminal hydrophobic motif of some AGC kinases like Akt and SGK. mTORC2 plays a crucial role in maintenance of normal and cancer cells through its association with ribosomes, and is involved in cellular metabolic regulation. Both complexes control each other as Akt regulates PRAS40 phosphorylation, which disinhibits mTORC1 activity, while S6K regulates Sin1 to modulate mTORC2 activity. Another significant component of mTORC2 is Sin1, which is crucial for mTORC2 complex formation and function. Allosteric inhibitors of mTOR, rapamycin and rapalogs, have essentially been ineffective in clinical trials of patients with GB due to their incomplete inhibition of mTORC1 or unexpected activation of mTOR via the loss of negative feedback loops. Novel ATP binding inhibitors of mTORC1 and mTORC2 suppress mTORC1 activity completely by total dephosphorylation of its downstream substrate pS6K

Dominguez-Brauer C, Khatun R, Elia AJ, et al.
E3 ubiquitin ligase Mule targets β-catenin under conditions of hyperactive Wnt signaling.
Proc Natl Acad Sci U S A. 2017; 114(7):E1148-E1157 [PubMed] Free Access to Full Article Related Publications
Wnt signaling, named after the secreted proteins that bind to cell surface receptors to activate the pathway, plays critical roles both in embryonic development and the maintenance of homeostasis in many adult tissues. Two particularly important cellular programs orchestrated by Wnt signaling are proliferation and stem cell self-renewal. Constitutive activation of the Wnt pathway resulting from mutation or improper modulation of pathway components contributes to cancer development in various tissues. Colon cancers frequently bear inactivating mutations of the adenomatous polyposis coli (

Mukherjee K, Sha X, Magimaidas A, et al.
Gadd45a deficiency accelerates BCR-ABL driven chronic myelogenous leukemia.
Oncotarget. 2017; 8(7):10809-10821 [PubMed] Free Access to Full Article Related Publications
The Gadd45a stress sensor gene is a member in the Gadd45 family of genes that includes Gadd45b & Gadd45g. To investigate the effect of GADD45A in the development of CML, syngeneic wild type lethally irradiated mice were reconstituted with either wild type or Gadd45a null myeloid progenitors transduced with a retroviral vector expressing the 210-kD BCR-ABL fusion oncoprotein. Loss of Gadd45a was observed to accelerate BCR-ABL driven CML resulting in the development of a more aggressive disease, a significantly shortened median mice survival time, and increased BCR-ABL expressing leukemic stem/progenitor cells (GFP+Lin- cKit+Sca+). GADD45A deficient progenitors expressing BCR-ABL exhibited increased proliferation and decreased apoptosis relative to WT counterparts, which was associated with enhanced PI3K-AKT-mTOR-4E-BP1 signaling, upregulation of p30C/EBPα expression, and hyper-activation of p38 and Stat5. Furthermore, Gadd45a expression in samples obtained from CML patients was upregulated in more indolent chronic phase CML samples and down regulated in aggressive accelerated phase CML and blast crisis CML. These results provide novel evidence that Gadd45a functions as a suppressor of BCR/ABL driven leukemia and may provide a unique prognostic marker of CML progression.

Chen W, Pan Y, Wang S, et al.
Cryptotanshinone activates AMPK-TSC2 axis leading to inhibition of mTORC1 signaling in cancer cells.
BMC Cancer. 2017; 17(1):34 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Cryptotanshinone (CPT), a fat-soluble phenanthraquinone from Salvia miltiorrhiza Bunge, has been demonstrated to inhibit phosphorylation of p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1), a couple of direct downstream effectors of the mammalian target of rapamycin complex 1 (mTORC1), resulting in cancer cell arrested in G0 phase and subsequent inhibition of proliferation. However, its concrete molecular mechanism about how CPT inhibits mTORC1 signaling pathway is unclear.
METHODS: one solution was used to check cell viability and western blotting for determining expression of the indicated proteins. Molecular docking was performed to assess the binding of CPT with mTOR. The co-immunoprecipitation assay was to analyze whether CPT could disrupt the mTORC1 and TSC1/TSC2 complex. Recombinant adenoviral dominant-negative AMPKα was used to downregulate expression of AMPKα and lentiviral AMPK and TSC2 to silence the AMPK and TSC2 in Rh30 cells.
RESULTS: Primarily, Rh30 cells expressing rapamycin-resistant mutant mTOR are also sensitive to CPT, while the molecular docking result for CPT binding to mTOR is negative, suggesting that CPT inhibition of mTORC1 is different from rapamycin. Then the related proteins of PTEN-PI3K pathway was proved not to be affected, but the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) was activated by a concentration- and time- dependent manner, meaning that it may be associated with AMPK. Further results indicated that compound C, inhibitor of AMPK, could clearly reversed CPT inhibitory effect on Rh30 cells, and dominant-negative AMPK in cancer cells conferred resistance to CPT inhibition of 4E-BP1 and phosphorylation of S6K1, as well as sh-AMPK. Furthermore, compared with AMPK-positive MEF cells, AMPK-negative MEF cells are less sensitive to CPT by the findings that 4E-BP1 and phosphorylation of S6K1 express comparatively more. Additionally, phosphorylation of tuberous sclerosis complex 2 (TSC2) was activated under the treatment of CPT, and down-expression of TSC2 by shRNA slightly recovered expression of 4E-BP1 and phosphorylation of S6K1, while co-immunoprecipitation of TSC2 did not alter expression of TSC1 by CPT.
CONCLUSION: CPT inhibiting mTORC1 pathway was mostly due to activation of AMPK-TSC2 axis rather than specific binding to mTORC1. CPT is a potent anticancer agent targeting AMPK.

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