IntS6 and the Integrator phosphatase module tune the efficiency of select premature transcription termination events.

The metazoan-specific Integrator complex catalyzes 3' end processing of small nuclear RNAs (snRNAs) and premature termination that attenuates the transcription of many protein-coding genes. Integrator has RNA endonuclease and protein phosphatase activities, but it remains unclear if both are required for complex function. Here, we show IntS6 (Integrator subunit 6) over-expression blocks Integrator function at a subset of Drosophila protein-coding genes, although having no effect on snRNAs or attenuation of other loci. Over-expressed IntS6 titrates protein phosphatase 2A (PP2A) subunits, thereby only affecting gene loci where phosphatase activity is necessary for Integrator function. IntS6 functions analogous to a PP2A regulatory B subunit as over-expression of canonical B subunits, which do not bind Integrator, is also sufficient to inhibit Integrator activity. These results show that the phosphatase module is critical at only a subset of Integrator-regulated genes and point to PP2A recruitment as a tunable step that modulates transcription termination efficiency.

Structural visualization of de novo transcription initiation by Saccharomyces cerevisiae RNA polymerase II.

Previous structural studies of the initiation-elongation transition of RNA polymerase II (pol II) transcription have relied on the use of synthetic oligonucleotides, often artificially discontinuous to capture pol II in the initiating state. Here, we report multiple structures of initiation complexes converted de novo from a 33-subunit yeast pre-initiation complex (PIC) through catalytic activities and subsequently stalled at different template positions. We determine that PICs in the initially transcribing complex (ITC) can synthesize a transcript of ∼26 nucleotides before transitioning to an elongation complex (EC) as determined by the loss of general transcription factors (GTFs). Unexpectedly, transition to an EC was greatly accelerated when an ITC encountered a downstream EC stalled at promoter proximal regions and resulted in a collided head-to-end dimeric EC complex. Our structural analysis reveals a dynamic state of TFIIH, the largest of GTFs, in PIC/ITC with distinct functional consequences at multiple steps on the pathway to elongation.

Influence of RNA circularity on Target RNA-Directed MicroRNA Degradation.

A subset of circular RNAs (circRNAs) and linear RNAs have been proposed to 'sponge' or block microRNA activity. Additionally, certain RNAs induce microRNA destruction through the process of Target RNA-Directed MicroRNA Degradation (TDMD), but whether both linear and circular transcripts are equivalent in driving TDMD is unknown. Here, we studied whether circular/linear topology of endogenous and artificial RNA targets affects TDMD. Consistent with previous knowledge that Cdr1as (ciRS-7) circular RNA protects miR-7 from Cyrano-mediated TDMD, we demonstrate that depletion of Cdr1as reduces miR-7 abundance. In contrast, overexpression of an artificial linear version of Cdr1as drives miR-7 degradation. Using plasmids that express a circRNA with minimal co-expressed cognate linear RNA, we show differential effects on TDMD that cannot be attributed to the nucleotide sequence, as the TDMD properties of a sequence often differ when in a circular versus linear form. By analysing RNA sequencing data of a neuron differentiation system, we further detect potential effects of circRNAs on microRNA stability. Our results support the view that RNA circularity influences TDMD, either enhancing or inhibiting it on specific microRNAs.

The capping enzyme facilitates promoter escape and assembly of a follow-on preinitiation complex for reinitiation.

After synthesis of a short nascent RNA, RNA polymerase II (pol II) dissociates general transcription factors (GTFs; TFIIA, TFIIB, TBP, TFIIE, TFIIF, and TFIIH) and escapes the promoter, but many of the mechanistic details of this process remain unclear. Here we developed an in vitro transcription system from the yeast Saccharomyces cerevisiae that allows conversion of the preinitiation complex (PIC) to bona fide initially transcribing complex (ITC), elongation complex (EC), and reinitiation complex (EC+ITC). By biochemically isolating postinitiation complexes stalled at different template positions, we have determined the timing of promoter escape and the composition of protein complexes associated with different lengths of RNA. Almost all of the postinitiation complexes retained the GTFs when pol II was stalled at position +27 relative to the transcription start site, whereas most complexes had completed promoter escape when stalled at +49. This indicates that GTFs remain associated with pol II much longer than previously expected. Nevertheless, the long-persisting transcription complex containing RNA and all of the GTFs is unstable and is susceptible to extensive backtracking of pol II. Addition of the capping enzyme and/or Spt4/5 significantly increased the frequency of promoter escape as well as assembly of a follow-on PIC at the promoter for reinitiation. These data indicate that elongation factors play an important role in promoter escape and that ejection of TFIIB from the RNA exit tunnel of pol II by the growing nascent RNA is not sufficient to complete promoter escape.

In vitro reconstitution of yeast RNA polymerase II transcription initiation with high efficiency.

Transcription initiation can be reconstituted from highly purified general transcription factors (GTFs), RNA polymerase II (pol II), and promoter DNA. However, earlier biochemical reconstitution systems had a serious technical limitation, namely very poor initiation efficiency. Due to the poor efficiency of the reaction and trace amounts of proteins involved in the pre-initiation complex (PIC) assembly, detection of transcription and PIC formation was only possible by the synthesis of a radiolabeled transcript and by immunoblotting for PIC components on templates. Here we describe a transcription system that is capable of initiating transcription with >90% efficiency of template usage using homogeneous, active yeast components including TFIIA, TFIIB, TBP, TFIIE, TFIIF, TFIIH, Sub1, and pol II. The abundant specifically assembled PICs on promoter DNA can be separated from free general transcription factors (GTFs) and pol II by density gradient sedimentation, irrespective of the length of promoter DNA. The system is robust, and can be modified to accommodate many other transcription factors, and the resulting complexes can be analyzed by SDS-PAGE followed by Coomassie Blue staining. This technical advance now paves the way to conduct definitive biochemical and structural studies of the complete process of pol II initiation from the PIC, through promoter escape, and finally to productive elongation.

CYP2D6 Allelic Variants *34, *17-2, *17-3, and *53 and a Thr309Ala Mutant Display Altered Kinetics and NADPH Coupling in Metabolism of Bufuralol and Dextromethorphan and Altered Susceptibility to Inactivation by SCH 66712.

Metabolic phenotype can be affected by multiple factors, including allelic variation and interactions with inhibitors. Human CYP2D6 is responsible for approximately 20% of cytochrome P450-mediated drug metabolism but consists of more than 100 known variants; several variants are commonly found in the population, whereas others are quite rare. Four CYP2D6 allelic variants-three with a series of mutations distal to the active site (*34, *17-2, *17-3) and one ultra-metabolizer with mutations near the active site (*53), along with reference *1 and an active site mutant of *1 (Thr309Ala)-were expressed, purified, and studied for interactions with the typical substrates dextromethorphan and bufuralol and the inactivator SCH 66712. We found that *34, *17-2, and *17-3 displayed reduced enzyme activity and NADPH coupling while producing the same metabolites as *1, suggesting a possible role for Arg296 in NADPH coupling. A higher-activity variant, *53, displayed similar NADPH coupling to *1 but was less susceptible to inactivation by SCH 66712. The Thr309Ala mutant showed similar activity to that of *1 but with greatly reduced NADPH coupling. Overall, these results suggest that kinetic and metabolic analysis of individual CYP2D6 variants is required to understand their possible contributions to variable drug response and the complexity of personalized medicine.

Multicellular Tumor Spheroids Combined with Mass Spectrometric Histone Analysis To Evaluate Epigenetic Drugs.

Multicellular tumor spheroids (MCTS) are valuable in vitro tumor models frequently used to evaluate the penetration and efficacy of therapeutics. In this study, we evaluated potential differences in epigenetic markers, i.e., histone post-translational modifications (PTMs), in the layers of the HCT116 colon carcinoma MCTS. Cells were grown in agarose-coated 96 well plates, forming reproducible 1-mm-diameter MCTS. The MCTS were fractionated into three radially concentric portions, generating samples containing cells from the core, the mid and the external layers. Using mass spectrometry (MS)-based proteomics and EpiProfile, we quantified hundreds of histone peptides in different modified forms; by combining the results of all experiments, we quantified the abundance of 258 differently modified peptides, finding significant differences in their relative abundance across layers. Among these differences, we detected higher amounts of the repressive mark H3K27me3 in the external layers, compared to the core. We then evaluated the epigenetic response of MCTS following UNC1999 treatment, a drug targeting the enzymes that catalyze H3K27me3, namely, the polycomb repressive complex 2 (PRC2) subunits enhancer of zeste 1 (EZH1) and enhancer of zeste 2 (EZH2). UNC1999 treatment resulted in significant differences in MCTS diameter under drug treatment of varying duration. Using matrix-assisted laser desorption/ionization (MALDI) imaging, we determined that the drug penetrates the entire MCTS. Proteomic analysis revealed a decrease in abundance of H3K27me3, compared to the untreated sample, as expected. Interestingly, we observed a comparable growth curve for MCTS under constant drug treatment over 13 days with those treated for only 4 days at the beginning of their growth. We thus demonstrate that MS-based proteomics can define significant differences in histone PTM patterns in submillimetric layers of three-dimensional (3D) cultures. Moreover, we show that our model is suitable for monitoring drug localization and regulation of histone PTMs after drug treatment.

Elaidic Acid, a Trans-Fatty Acid, Enhances the Metastasis of Colorectal Cancer Cells.

The effects of trans-fatty acids (TFAs) on cardiovascular disorders have been extensively studied, and the effect of TFAs on cancers has recently been recognized. This study examined the effects of elaidic acid (EA), a TFA, on colorectal cancer (CRC) progression. We demonstrated that EA enhanced the growth, survival, and invasion of the CRC cell lines, CT26, and HT29. Tumor growth and metastasis in the lung, liver, and peritoneum were significantly more enhanced in CRC cells treated with EA than those treated with the cis form of EA, oleic acid (OA), or vehicle. Spheres of CRC cells were formed at more pronounced numbers in EA-treated cells than in OA-treated cells. Compared to OA, EA treatment also induced expression of the stemness factors, nucleostemin, CD133, and Oct4. Moreover, spheres of EA-treated CRC cells were larger and more proliferative than spheres of OA-treated cells. Oral intake of EA also enhanced liver metastasis and CD133 expression of CRC cells in a dose-dependent manner. EA intake also increased resistance to 5-fluorouracil. Inhibition of Wnt and ERK1/2 abrogated EA-induced enhancement of metastasis. Our findings demonstrate that EA might provide prominent metastatic potential to CRC cells, which shows important implications for the treatment of CRC.

Multiplexed data independent acquisition (MSX-DIA) applied by high resolution mass spectrometry improves quantification quality for the analysis of histone peptides.

We present the MS-based application of the innovative, although scarcely exploited, multiplexed data-independent acquisition (MSX-DIA) for the analysis of histone PTMs. Histones are golden standard for complexity in MS based proteomics, due to their large number of combinatorial modifications, leading to isobaric peptides after proteolytic digestion. DIA has, thus, gained popularity for the purpose as it allows for MS/MS-based quantification without upfront assay development. In this work, we evaluated the performance of traditional DIA versus MSX-DIA in terms of MS/MS spectra quality, instrument scan rate and quantification precision using histones from HeLa cells. We used an MS/MS isolation window of 10 and 6 m/z for DIA and MSX-DIA, respectively. Four MS/MS scans were multiplexed for MSX-DIA. Despite MSX-DIA was programmed to perform two-fold more MS/MS events than traditional DIA, it acquired on average ∼5% more full MS scans, indicating even faster scan rate. Results highlighted an overall decrease of background ion signals using MSX-DIA, and we illustrated specific examples where peptides of different precursor masses were co-fragmented by DIA but not MSX-DIA. Taken together, MSX-DIA proved thus to be a more favorable method for histone analysis in data independent mode.

Expression of MAS1 in breast cancer.

MAS1 is a receptor for angiotensin 1-7 (A1-7), which is derived from angiotensin II (A-II) by the action of angiotensin converting enzyme (ACE) 2. MAS1 induces anti-A-II phenotypes, such as vessel dilation and depression of blood pressure. Using immunohistochemistry, we examined the role of MAS1 in 132 cases of invasive ductal carcinoma (IDC) of the breast. While benign mammary tissues expressed MAS1 at high levels, MAS1 expression was attenuated in all IDC, especially in scirrhous IDC. The decrease in MAS1 expression was associated with tumor growth, lymph node metastasis, and grade. MAS1 expression was inversely associated with the proliferation index and epidermal growth factor receptor and human epidermal growth factor receptor-2 expression. Of the 132 cases, 12 (9.1%) were triple-negative breast cancer (TNBC) cases. All TNBC cases (the 12 cases and the additional 36 cases using a tissue array) expressed MAS1. Using the TNBC cell lines 4T1 and MDA-MB-468, which expresses MAS1, we found that cell growth, anti-apoptotic survival and invasion were suppressed by MAS1 activation with A1-7 treatment and enhanced by MAS1 knockdown. In contrast, synergic effect was found between tamoxifen and A1-7 in a luminal A breast cancer cell line, MCF-7. Combination treatment with cisplatin, an ACE2 activator, and an A-II type 1 receptor blocker showed synergic effects on tumor growth inhibition of 4T1 tumors in a syngeneic mouse model. These findings suggest that MAS1 might act as an inhibitory regulator of breast cancer and may be a possible molecular target for this malignancy.

Cancer Therapeutic Effects of Titanium Dioxide Nanoparticles Are Associated with Oxidative Stress and Cytokine Induction.

Nanoparticles (NPs) are considered to influence the inflammatory process; however, the precise mechanism and the significance in tumors are still not clear. In this study, when CT26 and LL2 mouse cancer cells were treated with 6-nm anatase titanium dioxide NPs (TDNPs) without ultraviolet irradiation, oxidative stress and induction of inflammatory cytokines were observed. Oxidative stress was further increased by disease-associated conditions such as high glucose concentrations and hypoxia. Inhaled or orally administered TDNPs generated granulomatous lesions in the lungs and colon of the rodent models tested, with increased oxidative stress and inflammatory cytokines. Oxidative stress and inflammatory cytokines were also found in cancer cells treated with gold or carbon black NPs. Treatment of CT26 cells with 10- to 70-nm rutile TDNPs showed that smaller NPs produced more oxidative stress and inflammatory cytokines than larger ones did. To avoid diffusion of TDNPs and to minimize toxicity, 10-nm TDNPs were suspended in a collagen gel inserted into a subcutaneous tumor in a CT26 mouse. A single TDNP treatment via this method inhibited tumor growth in a size- and dose-dependent manner, and resulted in lower levels of urinary 8-OHdG when compared to systemically administered TDNPs. These findings suggest that TDNPs might be useful for the local treatment of tumors.

Mechanism-based inactivation of human cytochrome P450 3A4 by two piperazine-containing compounds.

Human cytochrome P450 3A4 (CYP3A4) is responsible for the metabolism of more than half of pharmaceutic drugs, and inactivation of CYP3A4 can lead to adverse drug-drug interactions. The substituted imidazole compounds 5-fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine (SCH 66712) and 1-[(2-ethyl-4-methyl-1H-imidazol-5-yl)methyl]-4-[4-(trifluoromethyl)-2-pyridinyl]piperazine (EMTPP) have been previously identified as mechanism-based inactivators (MBI) of CYP2D6. The present study shows that both SCH 66712 and EMTPP are also MBIs of CYP3A4. Inhibition of CYP3A4 by SCH 66712 and EMTPP was determined to be concentration, time, and NADPH dependent. In addition, inactivation of CYP3A4 by SCH 66712 was shown to be unaffected by the presence of electrophile scavengers. SCH 66712 displays type I binding to CYP3A4 with a spectral binding constant (Ks) of 42.9 ± 2.9 µM. The partition ratios for SCH 66712 and EMTPP were 11 and 94, respectively. Whole protein mass spectrum analysis revealed 1:1 binding stoichiometry of SCH 66712 and EMTPP to CYP3A4 and a mass increase consistent with adduction by the inactivators without addition of oxygen. Heme adduction was not apparent. Multiple mono-oxygenation products with each inactivator were observed; no other products were apparent. These are the first MBIs to be shown to be potent inactivators of both CYP2D6 and CYP3A4.

Serum CD10 is associated with liver metastasis in colorectal cancer.

INTRODUCTION: Colorectal cancer (CRC) is the third leading cause of cancer death in Japan. CD10 expression is closely associated with liver metastasis. In the present study, we explored the possibility of serum CD10 as a marker of liver metastasis in CRC. METHODOLOGY: BALB/c mouse with subcutaneous tumor of syngeneic CT26 CRC cells were examined serum CD10. In 84 CRC patients and patients undergoing hemodialysis, serum CD10 was examined. CD10 concentration was measured by enzyme-linked immunosorbent assay. RESULTS: In a mouse subcutaneous tumor model, serum CD10 correlated with the weight of the tumors. Serum CD10 was examined in 84 patients with CRC. The serum levels of CD10 were higher in patients with more advanced cancer stages. Patients with liver metastasis showed the highest levels of serum CD10 among all patients. Importantly, patients with high serum CD10 levels had metachronous liver metastasis. Healthy volunteers showed low levels of CD10; however, serum CD10 levels in patients undergoing hemodialysis showed levels as high as those with stage II and III CRC. A cutoff of serum CD10 set to >1000 pg/mL showed 70% sensitivity and 93% specificity for liver metastasis in CRC. This cutoff included all cases of metachronous liver metastasis. CONCLUSIONS: With the exclusion of mimicking factors, serum CD10 levels might serve as a useful marker of synchronous and metachronous liver metastasis in CRC.

Role of two types of angiotensin II receptors in colorectal carcinoma progression.

Angiotensin II (Ang-II) is a bioactive peptide associated closely with the progression and metastasis of colorectal cancer (CRC). We examined the expression and role of 2 Ang-II receptor types in 20 cases of CRC. Ang-II type 1 receptor (AT1R) protein was localized to the plasma membrane, whereas Ang-II type 2 receptor (AT2R) protein was localized to the nuclei. AT1R expression showed a direct correlation with tumor stage and liver metastasis, whereas AT2R expression showed an inverse correlation. A knockdown study of the AT1R or AT2R with Ang-II treatment was performed to reveal their individual roles in a mouse rectal cell line CMT93, which expresses both Ang-II receptor types. AT2R knockdown showed that the AT1R was associated with tumor growth, survival, invasion and VEGF-A secretion in CMT93 cells in a dose-dependent manner. In contrast, AT1R knockdown showed that the AT2R was associated with increased VEGF-A secretion at low Ang-II concentrations, whereas high concentrations of Ang-II inhibited tumor growth, survival, invasion and VEGF-A secretion. Thus, the AT1R showed a monophasic protumoral effect, while the AT2R showed a biphasic amphitumoral effect. Our findings suggest that a high angiotensinogen condition in the liver might evoke the antitumoral role of the AT2R in CRC cells.

Metoclopramide is metabolized by CYP2D6 and is a reversible inhibitor, but not inactivator, of CYP2D6.

1. Metoclopramide is a widely used clinical drug in a variety of medical settings with rare acute dystonic events reported. The aim of this study was to assess a previous report of inactivation of CYP2D6 by metoclopramide, to determine the contribution of various CYPs to metoclopramide metabolism, and to identify the mono-oxygenated products of metoclopramide metabolism. 2. Metoclopramide interacted with CYP2D6 with Type I binding and a Ks value of 9.56 ± 1.09 µM. CYP2D6 was the major metabolizer of metoclopramide and the two major products were N-deethylation of the diethyl amine and N-hydroxylation on the phenyl ring amine. CYPs 1A2, 2C9, 2C19, and 3A4 also metabolized metoclopramide. 3. While reversible inhibition of CYP2D6 was noted, CYP2D6 inactivation by metoclopramide was not observed under conditions of varying concentration or varying time using Supersomes(TM) or pooled human liver microsomes. 4. The major metabolites of metoclopramide were N-hydroxylation and N-deethylation formed most efficiently by CYP2D6 but also formed by all CYPs examined. Also, while metoclopramide is metabolized primarily by CYP2D6, it is not a mechanism-based inactivator of CYP2D6 in vitro.

Integrator-mediated clustering of poised RNA polymerase II synchronizes histone transcription.

Numerous components of the transcription machinery, including RNA polymerase II (Pol II), accumulate in regions of high local concentration known as clusters, which are thought to facilitate transcription. Using the histone locus of Drosophila nurse cells as a model, we find that Pol II forms long-lived, transcriptionally poised clusters distinct from liquid droplets, which contain unbound and paused Pol II. Depletion of the Integrator complex endonuclease module, but not its phosphatase module or Pol II pausing factors disperses these Pol II clusters. Consequently, histone transcription fails to reach peak levels during S-phase and aberrantly continues throughout the cell cycle. We propose that Pol II clustering is a regulatory step occurring near promoters that limits rapid gene activation to defined times. One Sentence Summary: Using the Drosophila histone locus as a model, we show that clustered RNA polymerase II is poised for synchronous activation.

Fatty acids inhibit anticancer effects of 5-fluorouracil in mouse cancer cell lines.

The present study investigated the effects of two major dietary fatty acid components, linoleic acid (LA) and elaidic acid (EA), on the antitumor effects of 5-fluorouracil (5-FU) in the LL2, CT26 and CMT93 mouse cancer cell lines. Concurrent treatment with LA and 5-FU elicited a decreased cell viability compared with treatment with 5-FU alone. In addition, increased inhibition of growth was observed following concurrent treatment with EA and 5-FU. Sequential treatment of LA followed by 5-FU abrogated the anticancer effects of 5-FU, and treatment with EA followed by 5-FU increased cancer cell growth in addition to abrogating the anticancer effects of 5-FU. The expression of the stem cell markers CD133 and nucleostemin (NS) increased in all three cell lines treated concurrently with 5-FU and either LA or EA when compared with cells treated with 5-FU alone. Aldehyde dehydrogenase activity in the cancer stem cells (CSCs), in response to concurrent treatment with 5-FU and either LA or EA, was increased compared with 5-FU treatment alone. 5-FU inhibited the growth of CT26 tumors, but co-treatment with either LA or EA abrogated this effect. NS-positive CSCs were more abundant in CT26 tumors treated with 5-FU and either LA or EA compared with those treated with 5-FU alone. The results of the present study suggested that, rather than altering the sensitivity of cancer cells to 5-FU, LA and EA may promote the survival of CSCs. The results indicated that dietary composition during chemotherapy is an important issue.

Differential quantification of isobaric phosphopeptides using data-independent acquisition mass spectrometry.

Phosphorylation is a post-translational modification (PTM) fundamental for processes such as signal transduction and enzyme activity. We propose to apply data-independent acquisition (DIA) using mass spectrometry (MS) to determine unexplored phosphorylation events on isobarically modified peptides. Such peptides are commonly not quantitatively discriminated in phosphoproteomics due to their identical mass.

NEDD 4 binding protein 2-like 1 promotes cancer cell invasion in oral squamous cell carcinoma.

Head and neck cancer, including oral squamous cell carcinoma, is the sixth most common cancer worldwide. Although cancer cell invasion and metastasis are crucial for tumor progression, detailed molecular mechanisms underlying the invasion and metastasis of oral squamous cell carcinoma are unclear. Comparison of transcriptional profiles using a cDNA microarray demonstrated that N4BP2L1, a novel oncogene expressed by neural precursor cells, is involved in oral squamous cell carcinoma. Expression of N4BP2L1 in oral squamous cell carcinoma is regulated by activation of miR-448 and is higher than in normal oral mucosa. Knockdown of N4BP2L1 and upregulation of miR-448 significantly reduced the invasive potential of oral squamous cell carcinoma cells. We studied N4BP2L1 expression in 187 cases of oral squamous cell carcinoma and found its overexpression to be significantly associated with nodal metastasis (P = 0.0155) and poor prognosis (P = 0.0136). Expression of miR-448 was found to be inversely associated with that of N4BP2L1 (P = 0.0019). Cox proportional hazards analysis identified N4BP2L1 expression as an independent predictor of disease-free survival (P = 0.0349). Our results suggest that N4BP2L1 plays an important role in tumor cell invasion in oral squamous cell carcinoma. Further studies on expression of N4BP2L1 may provide new insight into its function and clarify its potential as biomarker in human oral cancer.

Storkhead box 2 and melanoma inhibitory activity promote oral squamous cell carcinoma progression.

BACKGROUND: Storkhead box protein 2 (STOX2) is a transcriptional factor associated with pre-eclampsia with fetal growth restriction. We recently reported that melanoma inhibitory activity (MIA) promotes oral squamous cell carcinoma (OSCC) progression. However, the relationship between STOX2 and MIA remains unknown in malignancies. METHODS: We used immunohistochemistry and PCR to investigate MIA and STOX2 expression in OSCC. We also performed functional analysis in human OSCC cells. RESULTS: MIA and STOX2 mRNA levels were higher in OSCCs than in normal oral epithelial cells, and upregulation of STOX2 was significantly correlated with overexpression of MIA. Immunostaining for STOX2 was associated with nodal metastasis (P = 0.0002) and MIA expression (P < 0.0001). Furthermore, MIA expression (P = 0.0035) and STOX2 expression (P = 0.0061) were associated with poor outcome in OSCCs. In vitro analysis using OSCC cells revealed that MIA increased expression of STOX2 by paracrine manner. Moreover, STOX2 accelerated OSCC cell growth, invasion, suppressed apoptosis, and enhanced resistance to paclitaxel, cisplatin, and 5-FU. CONCLUSIONS: Our results suggest that MIA-STOX2 signaling may be a useful diagnostic and therapeutic target in OSCCs.