Migration And Invasion Inhibitory Protein Research Articles

MIIP downregulates PD-L1 expression through HDAC6 in cutaneous melanoma

PurposeImmune checkpoint inhibitors have improved the objective response rate and survival of melanoma patients. However, there are still many melanoma patients suffering from disease progression due to primary or secondary immune checkpoint inhibitor resistance, as is observed in the failure of anti-PD1/PD-L1 therapy. While the expression of valuable markers, such as TMB, MSI, and PD-L1, could serve as effective predictors of anti-checkpoint inhibitor therapies, tumor cell PD-L1 expression and its regulating mechanism would significantly affect the anti-PD-1 immunotherapy response and efficacy. Therefore, it is urgent to determine the function of PD-1/PD-L1 expression in melanoma and its associated pathways to enhance the efficacy of anti-PD-1 therapies.MethodsA cohort of 133 patients with histologically confirmed melanoma from Tianjin Medical University Cancer Institute & Hospital were included in this study. We performed immunohistochemical staining to detect the expression of Migration and invasion inhibitory protein (MIIP), HDAC6 and PD-L1. Kaplan–Meier and log-rank test were used for survival analysis. As for vitro, Western blot was used in melanoma cell lines to verify the signaling pathway that MIIP regulates PD-L1 expression.ResultsMIIP expression was decreased in melanoma and that the negative expression of MIIP was correlated with worse overall survival. The positive expression of HDAC6, a molecule that is downstream of MIIP, had a positive trend with decreased overall survival. At the same time, the positive expression of PD-L1, a crucial costimulatory molecule, was associated with decreased overall survival. Furthermore, there was a positive association between HDAC6 and PD-L1 protein expression (p < 0.01), and this correlation is more prominent in cutaneous melanoma than acral melanoma. In cutaneous melanoma cell lines, we found that increasing MIIP led to decreased HDAC6, pSTAT3, and PD-L1 expression. Knocking down MIIP led to increased HDAC6, pSTAT3, and PD-L1 expression. Combining the published results, showing that HDAC6 can regulate PD-L1 expression through STAT3, our present data suggest that MIIP inhibits the expression of PD-L1 by downregulating HDAC6 in melanoma. Most importantly, methods for targeting MIIP-HDAC6-PD-L1 pathways, such as treatment with HDAC6 inhibitors, might indicate a new therapeutic approach for enhancing immune checkpoint inhibitor therapies in melanoma.ConclusionsOur findings highlight the immunomodulatory effects of MIIP in the inhibition of PD-L1 expression by downregulating HDAC6 in melanoma. Methods for targeting MIIP-HDAC6-PD-L1 pathways might be new therapeutic approaches for enhancing immune checkpoint inhibitor therapies in melanoma.

MIIP downregulation drives colorectal cancer progression through inducing peri-cancerous adipose tissue browning

BackgroundThe enrichment of peri-cancerous adipose tissue is a distinctive feature of colorectal cancer (CRC), accelerating disease progression and worsening prognosis. The communication between tumor cells and adjacent adipocytes plays a crucial role in CRC advancement. However, the precise regulatory mechanisms are largely unknown. This study aims to explore the mechanism of migration and invasion inhibitory protein (MIIP) downregulation in the remodeling of tumor cell-adipocyte communication and its role in promoting CRC.ResultsMIIP expression was found to be decreased in CRC tissues and closely associated with adjacent adipocyte browning. In an in vitro co-culture model, adipocytes treated with MIIP-downregulated tumor supernatant exhibited aggravated browning and lipolysis. This finding was further confirmed in subcutaneously allografted mice co-injected with adipocytes and MIIP-downregulated murine CRC cells. Mechanistically, MIIP interacted with the critical lipid mobilization factor AZGP1 and regulated AZGP1’s glycosylation status by interfering with its association with STT3A. MIIP downregulation promoted N-glycosylation and over-secretion of AZGP1 in tumor cells. Subsequently, AZGP1 induced adipocyte browning and lipolysis through the cAMP-PKA pathway, releasing free fatty acids (FFAs) into the microenvironment. These FFAs served as the primary energy source, promoting CRC cell proliferation, invasion, and apoptosis resistance, accompanied by metabolic reprogramming. In a tumor-bearing mouse model, inhibition of β-adrenergic receptor or FFA uptake, combined with oxaliplatin, significantly improved therapeutic efficacy in CRC with abnormal MIIP expression.ConclusionsOur data demonstrate that MIIP plays a regulatory role in the communication between CRC and neighboring adipose tissue by regulating AZGP1 N-glycosylation and secretion. MIIP reduction leads to AZGP1 oversecretion, resulting in adipose browning-induced CRC rapid progression and poor prognosis. Inhibition of β-adrenergic receptor or FFA uptake, combined with oxaliplatin, may represent a promising therapeutic strategy for CRC with aberrant MIIP expression.

Inhibition of invasion and metastasis in choriocarcinoma by migration and invasion inhibitory protein

IntroductionChoriocarcinoma is a highly invasive gynaecologic malignancy. Molecular mechanism of metastasis in choriocarcinoma is poorly understood. Migration and invasion inhibitory protein (MIIP) regulates cell migration and invasion. Therefore, we aimed to elucidate the function of MIIP in choriocarcinoma. MethodsChoriocarcinoma cell lines, JAR and JEG-3, were transfected with lentivirus carrying the MIIP-interfering RNA (to downregulate MIIP expression) or left untransfected (negative control). Cell migration and invasion were studied using transwell migration assays and scratch assays. In vivo tumour burden was studied using tumour xenograft models in specific-pathogen-free nude mice and live imaging. We elucidated possible molecular signalling pathways using western blotting. ResultsIn transwell migration and scratch assays MIIP-downregulated JAR and JEG-3 cells migrated and invaded faster compared to their respective negative control cells. Migration and invasion by the MIIP-upregulated SWAN cells was slower than that by negative control SWAN cells. Live imaging revealed that bioluminescence values were higher in MIIP-downregulated tumours than in the negative control tumours. Mice with MIIP-downregulated tumours had higher serum human chorionic gonadotropin (HCG) levels than those with negative control tumours. The MIIP expression was negatively correlated with that of histone deacetylase (HDAC6) and positively correlated with that of acetylated α-tubulin. DiscussionThus, MIIP—by inhibiting cellular motility in choriocarcinoma—acts as a tumour suppressor gene. This highlights a potential therapeutic target for refractory choriocarcinoma. Additionally, HDAC6 and acetylated α-tubulin may be involved in the regulatory effects of MIIP on the biobehaviour of choriocarcinoma cells.

MIIP functions as a novel ligand for ITGB3 to inhibit angiogenesis and tumorigenesis of triple-negative breast cancer

Migration and invasion inhibitory protein (MIIP) has been identified as a tumor suppressor in various cancer types. Although MIIP is reported to exert tumor suppressive functions by repressing proliferation and metastasis of cancer cells, the detailed mechanism is poorly understood. In the present study, we found MIIP is a favorable indicator of prognosis in triple-negative breast cancer. MIIP could inhibit tumor angiogenesis, proliferation, and metastasis of triple-negative breast cancer cells in vivo and in vitro. Mechanistically, MIIP directly interacted with ITGB3 and suppressed its downstream signaling. As a result, β-catenin was reduced due to elevated ubiquitin-mediated degradation, leading to downregulated VEGFA production and epithelial mesenchymal transition. More importantly, we found RGD motif is essential for MIIP binding with ITGB3 and executing efficient tumor-suppressing effect. Our findings unravel a novel mechanism by which MIIP suppresses tumorigenesis in triple-negative breast cancer, and MIIP is thus a promising molecular biomarker or therapeutic target for the disease.

MIIP inhibits clear cell renal cell carcinoma proliferation and angiogenesis via negative modulation of the HIF-2α-CYR61 axis.

Objective:In various cancers, migration and invasion inhibitory protein (MIIP) is expressed at low level and is involved in cancer pathogenesis. Herein, we sought to explore the function of MIIP in clear cell renal cell carcinoma (ccRCC).Methods:CCK-8, colony formation, cell cycle, and endothelial cell tube formation assays were performed to evaluate the roles of MIIP in ccRCC proliferation and angiogenesis. To explore the underlying mechanism, we conducted RNA-sequencing, GSEA, qRT-PCR, Western blot, ELISA, cell transfection, coimmunoprecipitation, and ubiquitination assays in ccRCC cell lines. Furthermore, xenograft tumor growth in nude mice, and Ki-67 and CD31 staining in xenograft tissues were examined. Finally, the association of MIIP expression with clinical pathology and the expression status of HIF-2α and cysteine-rich 61 (CYR61) were further analyzed in human RCC tissues through Western blot and immunohistochemistry.Results:Both in vitro and in vivo functional experiments indicated that forced expression of MIIP inhibited ccRCC proliferation and angiogenesis, whereas silencing MIIP either in normal HK-2 cells or in ccRCC cells had the opposite effect (P < 0.05). Mechanistically, CYR61 was identified as a gene significantly downregulated by MIIP overexpression, and was required for the suppressive role of MIIP in ccRCC. MIIP was found to promote HSP90 acetylation and thus impair its chaperone function toward HIF-2α. Consequently, RACK1 binds HIF-2α and causes its ubiquitination and proteasomal degradation, thus decreasing the transcription of its target, CYR61. Finally, analyses of clinical samples demonstrated that MIIP is significantly downregulated in cancer vs. normal tissues in RCC cases, and its expression is negatively associated with histological grade, metastasis, the prognosis of patients with RCC, and the expression of HIF-2α and CYR61 (P < 0.05).Conclusions:MIIP is a novel tumor suppressor in ccRCC via negative regulation of HIF-2α-CYR61 axis.

MIIP inhibits the growth of prostate cancer via interaction with PP1\u03b1 and negative modulation of AKT signaling

BackgroundOver-activation of phosphatidylinositol 3-kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) signaling pathway is one of important mechanisms to promote castration resistant prostate cancer, the final stage of prostate cancer (PCa). Dysregulation of PP1-meditaed AKT dephosphorylation might contribute to such an event but is not fully understood. As a newly identified tumor suppressor, MIIP exerts its role in various types of cancer but has not been investigated in PCa.ResultsWe first demonstrated that overexpression of migration and invasion inhibitory protein (MIIP) in human PCa cell lines suppresses their growth while knockdown of MIIP does the opposite in vitro. Although MIIP has no effect on the expression of AR and its target genes or the nuclear translocation of AR in AR-positive PCa cells, MIIP overexpression significantly inhibits activation of AKT-mTOR pathway in both AR- positive and negative PCa cells whereas knockdown of MIIP enhances AKT-mTOR signaling. Using Western blot, immunofluorescence co-localization and co-immunoprecipitation analysis, we found that MIIP interacts with PP1α via its C-terminal part but does not affect its protein level. Importantly, silence of PP1α reversed the inhibitory effect of MIIP on AKT phosphorylation and cell growth in PCa cell lines, while MIIP∆C, which is incapable of interacting with PP1α, loses MIIP’s effect, suggesting that MIIP exerts its roles via interaction with PP1α. Further, MIIP overexpression inhibits the growth of both AR- positive and negative PCa xenograft in nude mice. Finally, immunohistochemical staining of PCa tissue microarray showed that MIIP expression level is downregulated in PCa and negatively correlated with Gleason score of PCa.ConclusionWe discovered that MIIP is a novel suppressor of oncogenic AKT-mTOR signaling in PCa by facilitating PP1-meditaed AKT dephosphorylation. Our study further emphasized the tumor suppressive role of MIIP and illustrated a novel mechanism.

Upregulation of MIIP regulates human breast cancer proliferation, invasion and migration by mediated by IGFBP2

AimsThe migration and invasion inhibitory protein (MIIP) was initially discovered in a yeast two-hybrid screen for proteins that interact and inhibit the migration and invasion-promoting protein insulin-like growth factor binding protein 2 (IGFBP2). This study aims to evaluate the biological effects of MIIP in breast cancer by targeting IGFBP2. Materials and methodsReverse transcription quantitative real-time polymerase chain reaction and Western blotting were used to evaluate the abnormal expression of MIIP and IGFBP2 in breast cancer tissue or breast cancer cell lines. Transfection assay was used to overexpress MIIP protein in breast cancer cells. MTT assay and colony formation assay were used to detect cell viability of breast cancer cells after MIIP overexpression. Transwell and wound-healing assays were used to detect cell invasion and migration after MIIP overexpression. ResultsMIIP was significantly decreased and IGFBP2 was significantly increased in breast cancer tissues versus para cancerous. Breast cancer tissues of HER2 overexpression and Basal-like were more significant than Luminal A and Luminal B. MIIP was obviously downregulated and IGFBP2 was upregulated in MDA-MB-231, SKBR3 and MCF-7 versus MCF-10A especially in MDA-MB-231. Cell proliferation, cell migration and cell invasion were significantly inhibited after overexpression of MIIP. IGFBP2 was downregulated after overexpression of MIIP. The effects of MIIP on cell proliferation, cell migration and invasion were significantly reversed by IGFBP2. ConclusionThe abnormal expression of MIIP in breast cancer affects the cell biological effects. IGFBP2 was regulated via MIIP which may be associated with these biological effects. These results reveal that MIIP can be a potential target for breast cancer treatment.

Long non‑coding RNA MALAT1 correlates with cell viability and mobility by targeting miR‑22‑3p in renal cell carcinoma via the PI3K/Akt pathway.

Renal cell carcinoma (RCC) is one of the most common types of cancer of the urinary tract in the world. Long non‑coding RNA MALAT1 (lncR‑MALAT1) is upregulated in RCC and is associated with the proliferation and migration of RCC. The present study aimed to investigate the regulating role of lncR‑MALAT1 in RCC as well as the possible underlying mechanisms. The relative expression of MALAT1 and miR‑22‑3p in RCC tumor tissues and cell lines was detected by qRT‑PCR. CCK‑8 and wound healing assay were used to evaluate cell proliferation and migration ability. Western blot analysis was used to detect the expression of Ki‑67, proliferating cell nuclear antigen (PCNA), matrix metalloproteinase‑3 (MMP‑3), migration and invasion inhibitory protein (MIIP), p‑PI3K and p‑Akt. The relationship between MALAT1 and miR‑22‑3p was examined by bioinformatic prediction analysis and luciferase reporter assay. Immunofluorescence was used to detect the activation of Akt. MALAT1 was highly expressed and the expression of miR‑22‑3p was suppressed in RCC tissues and cell lines. ShRNA‑mediated knockdown of MALAT1 significantly inhibited the viability and mobility of RCC cells invitro and invivo. Further experiments revealed that miR‑22‑3p was a target of MALAT1 and that miR‑22‑3p inhibitor abolished the effect of MALAT1 shRNA on cell proliferation, migration and inactivation of PI3K/AKT pathway. In conclusion, lncR‑MALAT1 affected the proliferation and migration of RCC cells by targeting miR‑22‑3p through the inactivation of the PI3K/Akt signaling pathway.

Expression and clinical significance of MIIP and PAK1 in endometrial carcinoma

Objective: To investigate the expressions of migration and invasion inhibitory protein (MIIP) and p21-activated kinase 1 (PAK1) in endometrial carcinoma (EC) and their correlation with clinicopathological features. Methods: The protein levels of MIIP and PAK1 in 135 paraffin-embedded EC tissues, 55 atypical hyperplasia of endometrium (AHE) and 88 normal endometrium (NE) tissues were quantified by immunohistochemistry, the clincial significance and the relationship of these two proteins were also analyzed. Results: The positive rates of MIIP expression in NE, AHE and EC tissues were 52.3%(46/88), 41.8% (23/55) and 34.8% (47/135), respectively. The expression of MIIP in EC was significantly lower than that of MIIP in NE (P<0.05). The positive rates of PAK1 expression in NE, AHE and EC tissues were 45.5% (40/88), 50.9% (28/55) and 62.2% (84/135), respectively. The expression of PAK1 in EC tissues was significantly higher than that of PAK1 in NE tissues (P<0.05). The expression of MIIP in EC tissues was significantly associated with myometrial invasion, International Federation of Gynaecology and Obstetrics (FIGO) stage and lymph node metastasis (P<0.05). The expression of PAK1 in EC tissues was significantly related with differentiation, myometrial invasion, FIGO stage and lymph node metastasis (P<0.05). The expressions of MIIP and PAK1 in EC tissues were marginally related with the overall survival of patients (P=0.092, P=0.052). The expression of MIIP in EC was negatively correlated with PAK1 (r=-0.329, P<0.001). Conclusions: The down-regulation of MIIP and up-regualtion of PAK1 paticipate in the initiation and development of EC, which are correlated with the poor prognosis of EC. The protein expression of MIIP is inversely related with PAK1 in EC.

PRP4 kinase induces actin rearrangement and epithelial-mesenchymal transition through modulation of the actin-binding protein cofilin

Cell actin cytoskeleton is primarily modulated by Rho family proteins. RhoA regulates several downstream targets, including Rho-associated protein kinase (ROCK), LIM-Kinase (LIMK), and cofilin. Pre-mRNA processing factor 4B (PRP4) modulates the actin cytoskeleton of cancer cells via RhoA activity inhibition. In this study, we discovered that PRP4 over-expression in HCT116 colon cancer cells induces cofilin dephosphorylation by inhibiting the Rho-ROCK-LIMK-cofilin pathway. Two-dimensional gel electrophoresis, and matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry (MALDI-TOF MS) analysis indicated increased expression of protein phosphatase 1A (PP1A) in PRP4-transfected HCT116 cells. The presence of PRP4 increased the expression of PP1A both at the mRNA and protein levels, which possibly activated cofilin through dephosphorylation and subsequently modulated the cell actin cytoskeleton. Furthermore, we found that PRP4 over-expression did not induce cofilin dephosphorylation in the presence of okadaic acid, a potent phosphatase inhibitor. Moreover, we discovered that PRP4 over-expression in HCT116 cells induced dephosphorylation of migration and invasion inhibitory protein (MIIP), and down-regulation of E-cadherin protein levels, which were further restored by the presence of okadaic acid. These findings indicate a possible molecular mechanism of PRP4-induced actin cytoskeleton remodeling and epithelial-mesenchymal transition, and make PRP4 an important target in colon cancer.

MIIP gene expression is associated with radiosensitivity in human nasopharyngeal carcinoma cells.

The present study aims to investigate the radiosensitization effect of the migration and invasion inhibitory protein (MIIP) gene on nasopharyngeal carcinoma (NPC) cells. The MIIP gene was transfected into NPC 5-8F and CNE2 cells. The level of MIIP was analyzed by quantitative reverse transcription-polymerase chain reaction analysis and western blot. The changes in radiosensitivity of the cells were analyzed by colony formation assay. The changes in cell apoptosis and cycle distribution following irradiation were detected by flow cytometry. The expression of BCL2 associated X, apoptosis regulator/B-cell lymphoma 2 was evaluated using western blot. DNA damage was analyzed by counting γ-H2AX foci. The expression levels of γ-H2AX were evaluated by immunofluorescence and western blot. In a previous study by the authors, the results indicated that the expression of MIIP gene evidently increased in MIIP-transfected 5-8F (5-8F OE) and MIIP-transfected CNE2 (CNE2 OE) cells compared with the parental or negative control cells. In the present study, the survival rate of 5-8F OE and CNE2 OE cells markedly decreased following irradiation (0, 2, 4, 6 and 8 Gy) compared with the negative control (5-8F NC and CNE2 NC) and the untreated (5-8F and CNE2) groups. The expression of MIIP was able to increase apoptosis, which resulted in G2/M cell cycle arrest and DNA damage repair was attenuated in 5-8F and CNE2 cells following irradiation as measured by the accumulation of γ-H2AX. It was indicated that MIIP expression is associated with the radiosensitivity of NPC cells and has a significant role in regulating cell radiosensitivity.

MiRNA-646-mediated reciprocal repression between HIF-1α and MIIP contributes to tumorigenesis of pancreatic cancer.

Migration and invasion inhibitory protein (MIIP) is recently identified as an inhibitor in tumor development. However, the regulatory mechanism and biological contributions of MIIP in pancreatic cancer (PC) have been not elucidated. In this study, we demonstrated a negative feedback of MIIP and hypoxia-induced factor-1α (HIF-1α), which was mediated by a hypoxia-induced microRNA. Compared with paracarcinoma tissues, MIIP was downregulated in PC tissues. Overexpression of MIIP significantly impeded the proliferation and invasion of PC cells both in vitro and in mouse xenograft models. We further verified MIIP was downregulated under hypoxia in a HIF-1α-mediated manner. Interestingly, although MIIP promoter containing two putative hypoxia response elements (HREs), the chromatin immunoprecipitation (ChIP) and luciferase reporter assays did not support an active interaction between HIF-1α and MIIP promoter. Meanwhile, microRNA array revealed a hypoxia-induced microRNA, miR-646, impaired stability of MIIP mRNA and consequently inhibited its expression by targeting the coding sequence (CDS). Coincidently, knockdown of miR-646 significantly repressed proliferation and invasion ability of PC cells both in vitro and in vivo by upregulating MIIP expression. Besides, ChIP and luciferase reporter assays further validated that HIF-1α activated transcription of miR-646 in hypoxia condition. Therefore, these results suggested HIF-1α indirectly regulated MIIP expression in post-transcriptional level through upregulating miR-646 transcription. Conversely, our results further revealed that MIIP suppressed deacetylase ability of histone deacetylase 6 (HDAC6) to promote the acetylation and degradation of HIF-1α, by which impairing HIF-1α accumulation. What is more, a specific relationship between downregulated MIIP and upregulated miR-646 expression was validated in PC samples. Moreover, the dysregulated miR-646 and MIIP expression was correlated with advanced tumor stage, lymphatic invasion, metastasis and shorter overall survival in PC patients. Together, our results highlight that the reciprocal loop of HIF-1α/miR-646/MIIP might be implemented as an applicable target for pancreatic cancer therapy.

PKC\u03b5 phosphorylates MIIP and promotes colorectal cancer metastasis through inhibition of RelA deacetylation

EGFR signaling is implicated in NF-κB activation. However, the concrete mechanisms by which the core transducer of NF-κB signaling pathway, RelA/p65 is regulated under EGFR activation remains to be further clarified. Here, we show that EGF stimulation induces PKCε-dependent phosphorylation of migration and invasion inhibitory protein (MIIP) at Ser303; this phosphorylation promotes the interaction between MIIP and RelA in the nucleus, by which MIIP prevents histone deacetylase 6 (HDAC6)-mediated RelA deacetylation, and thus enhances transcriptional activity of RelA and facilitates tumor metastasis. Meanwhile PP1, which functions as a phosphatase, is found to mediate MIIP-S303 dephosphorylation and its expression level inversely correlates with metastatic capability of tumor cells. Moreover, clinical analyses indicate the level of MIIP-S303 phosphorylation correlates with colorectal cancer (CRC) metastasis and prognosis. These findings uncover an unidentified mechanism underlying the precise regulation of NF-κB by EGF, and highlight the critical role of nuclear MIIP in tumor metastasis.

Expression of migration and invasion inhibitory protein (MIIP) in gastric adenocarcinoma and its clinicopathological significance

Objective To investigate the expression of migration and invasion inhibitory protein (MIIP) in gastric adenocarcinoma and its clinicopathological significance. Methods The expression of MIIP was detected in 90 samples of gastric adenocarcinoma and 90 samples of normal gastric tissues by tissue microarray and immunohistochemical method. The correlation of MIIP expression with clinicopathological parameters and prognosis of gastric adenocarcinoma patients was analyzed. Results The expression level of MIIP in gastric adenocarcinoma tissues was significantly lower than that in normal gastric tissues [30.0%(27/90)vs. 55.6%(50/90), χ2=12.006, P=0.001]. The down-regulated expression of MIIP in gastric adenocarcinoma tissues was significantly associated with larger tumor mass (χ2=4.116, P=0.042), higher T stage (χ2= 6.752, P=0.009 ), lymph nodes metastasis (χ2=7.234, P=0.007) and higher TNM stage (χ2=7.608, P=0.006). The 5-year overall survival rate was significantly higher in patients with high expression of MIIP than those with low expression (χ2=11.378, P=0.001). The tumor mass (OR=0.570, P=0.043), T stage (OR=0.213, P=0.036) and the expression level of MIIP (OR=1.967, P=0.042) were the independent predictors for prognosis of gastric adenocarcinoma. Conclusions The low expression level of MIIP is associated with poor prognosis in gastric adenocarcinomas. Key words: Gastric cancer; Adenocarcinoma; Prognosis; Migration and invasion inhibitory protein

GAS5 suppresses malignancy of human glioma stem cells via a miR-196a-5p/FOXO1 feedback loop

Glioma stem cells (GSCs) make up highly tumorigenic subpopulations within gliomas, and aberrant expression of GSC genes is a major underlying cause of glioma pathogenesis and treatment failure. The present study characterized the expression and function of long non-coding RNA growth arrest specific 5 (GAS5) in GSCs in order to elucidate the molecular mechanisms by which GAS5 contributes to glioma pathogenesis. We demonstrate that GAS5 suppresses GSC malignancy by binding to miR-196a-5p. miR-196a-5p, an onco-miRNA, stimulates GSC proliferation, migration, and invasion, in addition to reducing levels of apoptosis. miR-196a-5p specifically downregulates the expression of forkhead box protein O1 (FOXO1) by targeting its 3′ untranslated region (3′-UTR). FOXO1 upregulates expression of phosphotyrosine interaction domain containing 1 (PID1), thereby inhibiting GSC tumorigenicity and growth. FOXO1 also upregulates migration and invasion inhibitory protein (MIIP), resulting in attenuation of migration and invasion activities. Interestingly, we also show that FOXO1 promotes GAS5 transcription, thus forminga positive feedback loop. These data provide insights into potential new pathways for GSC molecular therapy and suggest that GAS5 may be an efficacious target for glioma treatments.

MIIP haploinsufficiency induces chromosomal instability and promotes tumour progression in colorectal cancer.

The gene encoding migration and invasion inhibitory protein (MIIP), located on 1p36.22, is a potential tumour suppressor gene in glioma. In this study, we aimed to explore the role and mechanism of action of MIIP in colorectal cancer (CRC). MIIP protein expression gradually decreased along the colorectal adenoma-carcinoma sequence and was negatively correlated with lymph node and distant metastasis in 526 colorectal tissue samples (p < 0.05 for all). Analysis of The Cancer Genome Atlas (TCGA) data showed that decreased MIIP expression was significantly associated with MIIP hemizygous deletion (p = 0.0005), which was detected in 27.7% (52/188) of CRC cases, and associated with lymph node and distant metastasis (p < 0.05 for both). We deleted one copy of the MIIP gene in HCT116 CRC cells using zinc finger nuclease technology and demonstrated that MIIP haploinsufficiency resulted in increased colony formation and cell migration and invasion, which was consistent with the results from siRNA-mediated MIIP knockdown in two CRC cell lines (p < 0.05 for all). Moreover, MIIP haploinsufficiency promoted CRC progression in vivo (p < 0.05). Genomic instability and spectral karyotyping assays demonstrated that MIIP haploinsufficiency induced chromosomal instability (CIN). Besides modulating the downstream proteins of APC/CCdc20 , securin and cyclin B1, MIIP haploinsufficiency inhibited topoisomerase II (Topo II) activity and induced chromosomal missegregation. Therefore, we report that MIIP is a novel potential tumour suppressor gene in CRC. Moreover, we characterized the MIIP gene as a novel CIN suppressor gene, through altering the stability of mitotic checkpoint proteins and disturbing Topo II activity. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

MIIP remodels Rac1-mediated cytoskeleton structure in suppression of endometrial cancer metastasis.

BackgroundEndometrial carcinoma (EC) is one of the most common malignancies of the female reproductive system. Migration and invasion inhibitory protein (MIIP) gene was recently discovered candidate tumor suppress gene which located at chromosome 1p36.22. 1p36 deletion was found in many types of tumor including EC. In the present study, we will determine the role and mechanism of MIIP in EC metastasis.MethodsImmunohistochemistry was used to measure MIIP expression in normal and EC tissue. Both gain-of-function (infection) and loss-of-function (siRNA) assays were used to alter MIIP expression levels. The effect of MIIP on cell migration and invasion was measured by transwell assay. F-actin immunofluorescence staining was used to observe the cell morphology. The activation of GTP-loaded Rac1 was evaluated by Rac activity assay kit. Immunoprecipitation/WB was used to measure the interaction between MIIP and PAK1.ResultsWe demonstrate that MIIP expression was significantly decreased in EC patients comparing to the normal ones, and decreased MIIP expression in EC tissues is associated with deep myometrial invasion, advanced stage, and the presence of lymph node metastasis. Using both gain-of-function (infection) and loss-of-function (siRNA) assays, we show that MIIP markedly blocked EC cell migration, whereas loss of MIIP led to increase in EC cell migration. We demonstrate that elevated expression of MIIP resulted in cytoskeleton reorganization with decreased formation of lamellipodia. We also provide evidence that MIIP is a key molecule in directing Rac1 signaling cascades in EC. Ectopically expressed MIIP consistently competed with Rac1-GTP for binding with the PAK1 p21-binding domain. Our data show that MIIP and PAK1 bind each other and that a C-terminal polyproline domain of MIIP is required for PAK1 binding. Deletion of the PAK1-binding domain of MIIP reduced cell migration-inhibiting activity.ConclusionsMIIP may function as a tumor suppressor gene for endometrial carcinoma. MIIP attenuates Rac1 signaling through a protein interaction network, and loss of this regulator may contribute to EC metastasis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-016-0342-6) contains supplementary material, which is available to authorized users.

MIIP expression predicts outcomes of surgically resected esophageal squamous cell carcinomas.

The migration and invasion inhibitory protein (MIIP) was shown to function as a tumor suppressor gene in gliomas by inhibiting tumor cell growth, migration, and invasion. However, its role and clinical significance in esophageal squamous cell carcinoma (ESCC) have not been elucidated. We investigated the correlation of MIIP expression and clinical outcome in a group of surgically resected ESCCs. Tissue microarrays constructed of 253 surgically resected ESCC primary tumors and paired paracancerous normal esophageal epithelia were used for MIIP evaluation by immunohistochemistry. The clinical and prognostic significance of MIIP expression was analyzed statistically. The expression of MIIP expression in cancer tissues was increased significantly in comparison with the paired paracancerous normal epithelia (P < 0.001). And, MIIP expression was associated with ESCC cells' differentiation (P < 0.001). By Kaplan-Meier analysis, patients with low MIIP expression exhibited significantly improved overall survival (OS, P = 0.039) and a tendency of improved disease-free survival (DFS, P = 0.086) than those with high MIIP expression. In addition, MIIP expression could distinguish OS or DFS of patients with tumors in stage T3-4 (P = 0.020, 0.028), N0 (P = 0.008, 0.032), and stage II (P = 0.004, 0.019), as well as at lower thoracic esophagus (P = 0.024, 0.090). Multivariate analysis showed that MIIP expression was an independent prognostic factor in ESCC OS and DFS. In conclusion, MIIP expressed higher in ESCCs than in paracancerous normal esophageal epithelia and was a positive, independent prognostic factor in resected ESCCs.

MIIP accelerates epidermal growth factor receptor protein turnover and attenuates proliferation in non-small cell lung cancer.

The migration and invasion inhibitory protein (MIIP) has been discovered recently to have inhibitory functions in cell proliferation and migration. Overexpression of MIIP reduced the intracellular steady-state level of epidermal growth factor receptor (EGFR) protein in lung cancer cells with no effect on EGFR mRNA expression compared to that in the control cells. This MIIP-promoted EGFR protein degradation was reversed by proteasome and lysosome inhibitors, suggesting the involvement of both proteasomal and lysosomal pathways in this degradation. This finding was further validated by pulse-chase experiments using 35S-methionine metabolic labeling. We found that MIIP accelerates EGFR protein turnover via proteasomal degradation in the endoplasmic reticulum and then via the lysosomal pathway after its entry into endocytic trafficking. MIIP-stimulated downregulation of EGFR inhibits downstream activation of Ras and blocks the MEK signal transduction pathway, resulting in inhibition of cell proliferation. The negative correlation between MIIP and EGFR protein expression was validated in lung adenocarcinoma samples. Furthermore, the higher MIIP protein expression predicts a better overall survival of Stage IA-IIIA lung adenocarcinoma patients who underwent radical surgery. These findings reveal a new mechanism by which MIIP inhibits cell proliferation.

Altered expression and loss of heterozygosity of the migration and invasion inhibitory protein (MIIP) gene in breast cancer.

Previous studies have characterized the migration and invasion inhibitory protein (MIIP) as a novel putative tumor-suppressor gene that regulates cell migration and invasion as well as the mitotic checkpoint. The MIIP gene is located on chromosome 1p36.22, a common site for deletion in many solid tumors including breast cancer. In the present study, we evaluated MIIP expression and allelic deletion to gain insight into the role of the MIIP gene in breast cancer. MIIP gene mRNA and protein expression was assessed in 86 matched breast cancer and adjacent normal tissues. Loss of heterogeneity (LOH) of the MIIP gene was determined using single-nucleotide polymorphism (SNP) and microsatellite (MS) markers in 149 breast carcinomas and the corresponding normal lymphocytes. The analysis revealed that the expression levels of MIIP mRNA and protein were downregulated in tumor specimens compared to those in corresponding adjacent tissues. Advanced clinical stage and tumor size >2 cm were associated with a decreased MIIP expression level. Twenty-six percent (37/142) of tumors were shown to have LOH at the MIIP locus by MS and SNP markers. Breast cancer patients with LOH at the SNP marker rs2295283 experienced shorter survival time. The attenuated expression and LOH of the MIIP gene may contribute to the poor prognosis of breast cancer, supporting a tumor-suppressing role of MIIP gene in the pathogenesis of this disease.