M-phase Phosphoprotein Research Articles

MiR‑576‑3p/M‑phase phosphoprotein 8 axis regulates the malignant progression of hepatocellular carcinoma cells via the PI3K/Akt signaling pathway.

Hepatocellular carcinoma (HCC) is a fatal digestive system cancer with unclear pathogenesis. M-phase phosphoprotein 8 (MPP8) has been shown to play a vital role in several cancer types, such as non-small cell lung cancer, gastric cancer and melanoma; however, there have been no studies into its role in HCC. The present study aimed to evaluate the role of MPP8 in regulating malignant phenotypes of liver cancer cells, and to further investigate the underlying mechanism. Bioinformatics analysis was performed to analyze related data from a public database, and to predict the potential microRNAs (miRNAs) that might target MPP8 mRNA; reverse transcription-quantitative PCR was used to measure the levels of mRNA and miRNA; western blotting was employed to detect protein levels; Cell Counting Kit-8 (CCK-8) and plate colony formation assays, wound healing assay and Transwell invasion assay were performed to evaluate the ability of cell proliferation, migration and invasion, respectively; dual-luciferase reporter gene assay was performed to identify the target association. The results showed that MPP8 was a risk factor for the survival of patients with HCC, and was up-regulated in HCC tissue samples and cell lines; MPP8 knockdown inhibited the proliferation, migration and invasion of liver cancer cells; MPP8 knockdown suppressed the PI3K/Akt pathway, and activation of this pathway reversed the inhibited liver cancer cell phenotypes by down-regulating MPP8; miR-576-3p, which was low in liver cancer cells, negatively regulated MPP8 expression by directly targeting its mRNA; up-regulating MPP8 expression reversed the inhibited signaling pathway and malignant phenotypes of liver cancer cells by miR-576-3p overexpression. In conclusion, the miR-576-3p/MPP8 axis regulates the proliferation, migration, and invasion of liver cancer cells through the PI3K/Akt signaling pathway. These findings lead novel insights into HCC progression, and propose MPP8 as a potential therapeutic target for HCC.

Optogenetics for sensors: On-demand fluorescent labeling of histone epigenetics

Post-translational modifications of histones to a large extent determine the functional state of chromatin loci. Dynamic visualization of histone modifications with genetically encoded fluorescent sensors makes it possible to monitor changes in the epigenetic state of a single living cell. At the same time, the sensors can potentially compete with endogenous factors recognizing these modifications. Thus, prolonged binding of the sensors to chromatin can affect normal epigenetic regulation. Here, we report an optogenetic sensor for live-cell visualization of histone H3 methylated at lysine-9 (H3K9me3) named MPP8-LAMS (MPP8-based light-activated modification sensor). MPP8-LAMS consists of several fusion protein parts (from N- to C-terminus): i) nuclear export signal (NES), ii) far-red fluorescent protein Katushka, iii) H3K9me3-binding reader domain of the human M phase phosphoprotein 8 (MPP8), iv) the light-responsive AsLOV2 domain, which exposes a nuclear localization signal (NLS) upon blue light stimulation. In the dark, due to the NES, MPP8-LAMS is localized in the cytosol. Under blue light illumination, MPP8-LAMS underwent an efficient translocation from cytosol to nucleus, enabling visualization of H3K9me3-enriched loci. Such an on-demand visualization minimizes potential impact on cell physiology as most of the time the sensor is separated from its target. In general, the present work extends the application of optogenetics to the area of advanced use of genetically encoded sensors.

Retraction Statement: Lentivirus-mediated knockdown of Mphase phosphoprotein 8 inhibits proliferation of colon cancer cells.

Retraction Statement: Lentivirus-mediated knockdown of Mphase phosphoprotein 8 inhibits proliferation of colon cancer cells.

Exercise intervention during pregnancy induces DNA methylation alterations in maternal blood and cord blood.

Exercise intervention during pregnancy induces DNA methylation alterations in maternal blood and cord blood.

Characterization of Hepatoma-Derived Growth Factor-Related Protein 2 Interactions with Heterochromatin.

The expression of genetic information is tightly controlled by chromatin regulatory proteins, including those in the heterochromatin gene repression family. Many of these regulatory proteins work together on the chromatin substrate to precisely regulate gene expression during mammalian development, giving rise to many different tissues in higher organisms from a fixed genomic template. Here we identify and characterize the interactions of two related heterochromatin regulatory proteins, heterochromatin protein 1 alpha (HP1α) and M-phase phosphoprotein 8 (MPP8), with hepatoma-derived growth factor-related protein 2 (HRP2). We find in biochemical experiments that HRP2 copurifies and co-sediments with heterochromatin-associated proteins, including HP1α and MPP8. Using the Chromatin in vivo Assay in multiple cell types, we demonstrate that HP1α-mediated gene repression dynamics are altered by the presence of HRP2. Furthermore, the knockout of HRP2 in MDA-MB-231 cells results in significant changes to chromatin structure and stability, which alter gene expression patterns. Here, we detail a mechanism by which HRP2 contributes to epigenetic transcriptional regulation through engagement with heterochromatin-associated proteins to stabilize the chromatin landscape and influence gene expression.

The HUSH complex controls brain architecture and protocadherin fidelity

The HUSH (human silencing hub) complex contains the H3K9me3 binding protein M-phase phosphoprotein 8 (MPP8) and recruits the histone methyltransferase SETDB1 as well as Microrchidia CW-type zinc finger protein 2 (MORC2). Functional and mechanistic studies of the HUSH complex have hitherto been centered around SETDB1 while the in vivo functions of MPP8 and MORC2 remain elusive. Here, we show that genetic inactivation of Mphosph8 or Morc2a in the nervous system of mice leads to increased brain size, altered brain architecture, and behavioral changes. Mechanistically, in both mouse brains and human cerebral organoids, MPP8 and MORC2 suppress the repetitive-like protocadherin gene cluster in an H3K9me3-dependent manner. Our data identify MPP8 and MORC2, previously linked to silencing of repetitive elements via the HUSH complex, as key epigenetic regulators of protocadherin expression in the nervous system and thereby brain development and neuronal individuality in mice and humans.

A Peptidomimetic Ligand Targeting the Chromodomain of MPP8 Reveals HRP2's Association with the HUSH Complex.

The interpretation of histone post-translational modifications (PTMs), specifically lysine methylation, by specific classes of "reader" proteins marks an important aspect of epigenetic control of gene expression. Methyl-lysine (Kme) readers often regulate gene expression patterns through the recognition of a specific Kme PTM while participating in or recruiting large protein complexes that contain enzymatic or chromatin remodeling activity. Understanding the composition of these Kme-reader-containing protein complexes can serve to further our understanding of the biological roles of Kme readers, while small molecule chemical tools can be valuable reagents in interrogating novel protein-protein interactions. Here, we describe our efforts to target the chromodomain of M-phase phosphoprotein 8 (MPP8), a member of the human silencing hub (HUSH) complex and a histone 3 lysine 9 trimethyl (H3K9me3) reader that is vital for heterochromatin formation and has specific roles in cancer metastasis. Utilizing a one-bead, one-compound (OBOC) combinatorial screening approach, we identified UNC5246, a peptidomimetic ligand capable of interacting with the MPP8 chromodomain in the context of the HUSH complex. Additionally, a biotinylated derivative of UNC5246 facilitated chemoproteomics studies which revealed hepatoma-derived growth factor-related protein 2 (HRP2) as a novel protein associated with MPP8. HRP2 was further shown to colocalize with MPP8 at the E-cadherin gene locus, suggesting a possible role in cancer cell plasticity.

MPP8 Promotes Proliferation and Restrains Apoptosis in Osteosarcoma by Regulating p38αMAPK Pathway.

Osteosarcoma is the most common primary bone malignancy. We aim to investigate that role of M-phase phosphoprotein 8 (MPP8) on proliferation and apoptosis in osteosarcoma. Briefly, the current research reported an in vitro study investigating the role MPP8 in OS tumorigenesis. Consequently, we found that the MPP8 expression was upregulated in osteosarcoma tissues and in osteosarcoma cell lines. Interestingly, MPP8 knockdown via shRNA restrained the cell viability and proliferation of U2OS and Saos-2 cells. In addition, MPP8 knockdown promoted the apoptosis of U2OS and Saos-2 cells, while MPP8 overexpression promotes proliferation and inhibited the cell apoptosis of osteosarcoma cells. These results suggested that MPP8 may serve as a contributor for osteosarcoma growth and inhibition of MPP8 may help restrain the development of osteosarcoma. Importantly, we found that MPP8 overexpression suppressed the protein levels of HOXA5, p38αMAPK, increased cell proliferation and inhibited cell apoptosis, while co-transfection with HOXA5 overexpression suppressed the cell proliferation and increased cell apoptosis. These results indicated that MPP8 contributed to cell proliferation and the underlying mechanism might be involved with HOXA5/ p38αMAPK pathway.

Zinc finger C3H1 domain-containing protein (ZFC3H1) evaluates the prognosis and treatment of prostate adenocarcinoma (PRAD): A study based on TCGA data

ABSTRACT The present study was aimed to evaluate the expression profile of Zinc finger C3H1 domain-containing protein (ZFC3H1) using bioinformatic analysis of public datasets from The Cancer Genome Atlas database (TCGA). The results showed that the expression levels of ZFC3H1 were notably lower than the corresponding non-cancerous tissues in prostate adenocarcinoma (PRAD), and patients in the high ZFC3H1-expression group showed poor survival. We hypothesized that the low expression of ZFC3H1 in tumor tissue might have be an inhibitory effect on the autoimmune system. We predicted the regulatory target and protein interaction partner network of ZFC3H1, and identified a PPI network composed of 26 node genes in PRAD. Furthermore, we found that the expression levels of MPHOSPH6 (encoding M-phase phosphoprotein 6) and MRPS31 (encoding mitochondrial ribosomal protein S31) were lower in PRAD tissues than in non-cancerous tissues, and the survival time of patients with high MPHOSPH6 and MRPS31 expression was poor. To further demonstrate the role of ZC3H1 in PRAD, we knocked-down the ZFC3H1 expression and found that the inhibition of ZFC3H1 significantly inhibited PRAD cell migration and invasion. Furthermore, ZFC3H1 siRNA treatment could reduce cell viability and increase the number of apoptotic cells in PRAD cells. Taken together, ZFC3H1 could represent a new marker for PRAD prognosis and provide a reference for the development of new therapies to treat PRAD.

M-Phase Phosphoprotein 9 upregulation associates with poor prognosis and activates mTOR signaling in gastric cancer.

The function of M-Phase Phosphoprotein 9 (MPHOSPH9) has not been investigated in gastric cancer yet. In the present study, the public cancer databases Oncomine and TCGA were analyzed, and MPHOSPH9 was found upregulated in gastric cancer tumor tissues. Immunohistochemistry (IHC) was also carried out to further confirm the results, and IHC analysis showed MPHOSPH9 was elevated in tumor tissues compared with the paracancerous tissues. QRT-PCR analysis also revealed that MPHOSPH9 mRNA was upregulated in gastric cancer cell lines. In addition, Kaplan-Meier estimates showed gastric cancer patients with high MPHOSPH9 level predicted a poor prognosis. Then, Western blot and CCK-8 assay showed overexpressed MPHOSPH9 enhanced gastric cancer cell proliferation, but MPHOSPH9 knockdown suppressed gastric cancer cell proliferation. Additionally, Western blot showed that MPHOSPH9 regulated the activation of mTOR, and overexpressed MPHOSPH9 reduced the inhibitory effects of mTOR inhibitors on cell survival in gastric cancer cells. Taken together, our results suggested that MPHOSPH9 .could be an oncogene in gastric cancer by regulating mTOR signaling.

The 5.8S pre-rRNA maturation factor, M-phase phosphoprotein 6, is a female fertility factor required for oocyte quality and meiosis.

ObjectivesM‐phase phosphoprotein 6 (MPP6) is important for 5.8S pre‐rRNA maturation in somatic cells and was screened as a female fertility factor. However, whether MPP6 functions in oocyte meiosis and fertility is not yet known. We aimed to address this.Materials and MethodsMouse oocytes with surrounded nucleus (SN) or non‐surrounded nucleus (NSN) were used for all experiments. Peptide nanoparticle‐mediated antibody transfection was used to deplete MPP6. Immunofluorescence staining, immunohistochemistry and live tracker staining were used to examine MPP6 localization and characterize phenotypes after control or MPP6 depletion. High‐fidelity PCR and fluorescence in situ hybridization (FISH) were used to examine the localization and level of 5.8S rRNAs. Western blot was used to examine the protein level. MPP6‐EGFP mRNA microinjection was used to do the rescue.ResultsMPP6 was enriched within ovaries and oocytes. MPP6 depletion significantly impeded oocyte meiosis. MPP6 depletion increased 5.8S pre‐rRNA. The mRNA levels of MPP6 and 5.8S rRNA decreased within ageing oocytes, and MPP6 mRNA injection partially increased 5.8S rRNA maturation and improved oocyte quality.ConclusionsMPP6 is required for 5.8S rRNA maturation, meiosis and quality control in mouse oocytes, and MPP6 level might be a marker for oocyte quality.

M-phase phosphoprotein 8 promotes gastric cancer growth and metastasis via p53/Bcl-2 and EMT-related signaling pathways.

The main issue of this study is to demonstrate whether M-phase phosphoprotein 8 (MPP8) affect gastric tumor growth and metastasis. Retrospective study was proceeded in 280 patients' surgical specimens with different disease stages. Loss-of-function assays, including 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, flow cytometry, and transwell assays were performed to evaluate the biological function of MPP8 in gastric cancer cells. Apoptosis and metastasis relative biomarkers were measured by quantitative real-time polymerase chain reaction and Western blot analysis. Compared with normal adjacent tissues, obviously elevated MPP8 expression was found in gastric cancer tissues. Elevated MPP8 expression was associated with male sex (vs female sex), intermediate differentiation (vs poorly differentiated cancer), and later stage (vs earlier stage). Furthermore, MPP8 overexpression in tumor tissues was marginally associated with a poor prognosis, with a significant relationship between MPP8 overexpression and prognosis among patients with poorly differentiated gastric cancer. Inhibition of MPP8 in these cells significantly suppressed proliferation and colony formation, promoted apoptosis, and repressed invasion. Furthermore, silencing of MPP8 remarkably increased apoptosis-related proteins (p53, Bax, and PARP) expression, but downregulated Bcl-2 expression. Silencing of MPP8 also decreased the expression of metastasis pathway-related proteins (N-cadherin and vimentin), and as well as the levels of anti-oncogene ZEB1, MET, and KRAS mRNA. Our findings demonstrated that MPP8 might be an oncogene by positively regulating gastric cancer cell function through the p53/Bcl-2 and epithelial to mesenchymal transition-related signaling pathways.

169P - M-phase phosphoprotein 8 promotes gastric cancer growth and metastasis via p53/Bcl-2 and EMT-related signalling pathways

BackgroundThe main issue of this study is to demonstrate whether M-phase phosphoprotein 8 (MPP8) affect gastric tumour growth and metastasis. MethodsRetrospective study was proceeded in 280 patients’ surgical specimens with different disease stages. Lentivirus delivery of short hairpin RNA targeting MPP8 was also used to explore the function of MPP8 in gastric cancer cells. Quantitative real-time PCR and western blot were applied to measure apoptosis and metastasis relative biomarker. ResultsThe present study revealed that MPP8 expression was higher in surgical specimens from patients with gastric cancer (vs. normal adjacent tissues), and that elevated expression was associated with male sex (vs. female sex), intermediate differentiation (vs. poorly differentiated cancer), and later stage (vs. earlier stage). Furthermore, MPP8 overexpression in tumour tissues was marginally associated with a poor prognosis, with a significant relationship between MPP8 overexpression and prognosis among patients with poorly differentiated gastric cancer. Inhibition of MPP8 in these cells significantly suppressed proliferation and colony formation, promoted apoptosis, and repressed invasion. Furthermore, silencing of MPP8 remarkably increased the expression of apoptosis-related proteins (p53, Bax, and PARP), and down-regulated Bcl-2 expression. Silencing of MPP8 also decreased the expression of metastasis pathway-related proteins (N-cadherin and vimentin), and as well as the levels of anti-oncogene ZEB1, MET, and KRAS mRNA. ConclusionsThese results suggest that MPP8 plays a critical role in regulating gastric cancer apoptosis and metastasis via the p53/Bcl-2 and EMT-related signalling pathways, respectively, which may indicate that MPP8 is a promising target for treating gastric cancer. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

The H3K9 Methylation Writer SETDB1 and its Reader MPP8 Cooperate to Silence Satellite DNA Repeats in Mouse Embryonic Stem Cells.

SETDB1 (SET Domain Bifurcated histone lysine methyltransferase 1) is a key lysine methyltransferase (KMT) required in embryonic stem cells (ESCs), where it silences transposable elements and DNA repeats via histone H3 lysine 9 tri-methylation (H3K9me3), independently of DNA methylation. The H3K9 methylation reader M-Phase Phosphoprotein 8 (MPP8) is highly expressed in ESCs and germline cells. Although evidence of a cooperation between H3K9 KMTs and MPP8 in committed cells has emerged, the interplay between H3K9 methylation writers and MPP8 in ESCs remains elusive. Here, we show that MPP8 interacts physically and functionally with SETDB1 in ESCs. Indeed, combining biochemical, transcriptomic and genomic analyses, we found that MPP8 and SETDB1 co-regulate a significant number of common genomic targets, especially the DNA satellite repeats. Together, our data point to a model in which the silencing of a class of repeated sequences in ESCs involves the cooperation between the H3K9 methylation writer SETDB1 and its reader MPP8.

A Monoclonal Antibody to M-Phase Phosphoprotein 1/Kinesin-Like Protein KIF20B.

Kinesin-like protein KIF20B, originally named M-phase phosphoprotein 1 (MPP1), is a plus-end-directed kinesin-related protein that exhibits in vitro microtubule-binding and -bundling properties as well as microtubule-stimulated ATPase activity. It has been characterized as a slow molecular motor that moves toward the plus-end of microtubules. Human autoantibodies directed against KIF20B have been described in up to 25% of patients with idiopathic ataxia and less commonly in other neuropathies and autoinflammatory conditions. One of the limitations of research into the structure and function of KIF20B has been a reliable monoclonal antibody that can be used in a variety of applications. To establish a reference standard for anti-KIF20B immunoassays and facilitate studies on the role of KIF20B in developmental cell biology, we developed an IgG1 monoclonal antibody, 10C7, which reacts with the cognate KIF20B protein in Western immunoblots and in addressable laser bead immunoassays. In HEp2 cells, leptomeningeal pericytes, and transfected HEK293T cells, indirect immunofluorescence studies showed that reactivity was mainly localized to a proportion of interphase nuclei, but during metaphase, it was redistributed throughout the cytoplasm and perichromatin mass. Later in telophase/anaphase, KIF20B was localized to the stem body and midzone of the midbody. 10C7 also showed remarkable staining of a subset of cells in the cerebellum, ovary, and testis tissues. KIF20B was shown to have extensive coiled-coil domains. The monoclonal antibody, 10C7, will be of value to diagnostic laboratory scientists interested in having a reliable reference standard for anti-KIF20B immunoassays as well as cell, molecular, and developmental biology researchers.

KIF20B promotes the progression of clear cell renal cell carcinoma by stimulating cell proliferation.

Renal cell carcinoma (RCC) is a common urinary system cancer with high morbidity and mortality rate. Clear cell renal cell carcinoma (ccRCC) is a highly aggressive and common type of RCC. More and effective therapeutic targets are badly needed for the treatment of ccRCC. Kinesin family protein (KIF)20B, also named M-phase phosphoprotein 1, was reported as a microtubule-associated, plus-end-directed kinesin. KIF20B was involved in multiple cellular processes such as cytokinesis. Multiple studies indicated the oncogenic role for KIF20B in several types of tumors, including breast cancer and bladder cancer. However, the possible role of KIF20B in the progression of renal carcinoma is still unknown. Herein, our study demonstrated that KIF20B was relatively highly expressed in ccRCC tissues. In addition, KIF20B was inversely related to the clinical features including tumor size and T stage. We further found that inhibition of the KIF20B expression by a specific short hairpin RNA obviously reduces proliferation of ccRCC cells both in vitro and in vivo. Our study reveals the involvement of KIF20B in ccRCC progression. Generally, KIF20B is a promising novel therapeutic for the treatment of clear cell RCC.

M-Phase Phosphoprotein 9 regulates ciliogenesis by modulating CP110-CEP97 complex localization at the mother centriole

The primary cilium is elongated from the mother centriole and has diverse signaling roles during development and disease. The CP110-CEP97 complex functions as a negative regulator of ciliogenesis, although the mechanisms regulating its mother centriole localization are poorly understood. Here we show that M-Phase Phosphoprotein 9 (MPP9) is recruited by Kinesin Family Member 24 (KIF24) to the distal end of mother centriole where it forms a ring-like structure and recruits CP110-CEP97 by directly binding CEP97. Loss of MPP9 causes abnormal primary cilia formation in growing cells and mouse kidneys. After phosphorylation by Tau Tubulin Kinase 2 (TTBK2) at the beginning of ciliogenesis, MPP9 is targeted for degradation via the ubiquitin-proteasome system, which facilitates the removal of CP110 and CEP97 from the distal end of the mother centriole. Thus, MPP9 acts as a regulator of ciliogenesis by regulating the localization of CP110-CEP97 at the mother centriole.

Tri-methylation of ATF7IP by G9a/GLP recruits the chromodomain protein MPP8

BackgroundG9a and the related enzyme GLP were originally identified as histone lysine methyltransferases and then shown to also methylate several other non-histone proteins.ResultsHere, we performed a comprehensive screen to identify their substrates in mouse embryonic stem cells (mESCs). We identified 59 proteins, including histones and other known substrates. One of the identified substrates, activating transcriptional factor 7-interacting protein 1 (ATF7IP), is tri-methylated at a histone H3 lysine 9 (H3K9)-like mimic by the G9a/GLP complex, although this complex mainly introduces di-methylation on H3K9 and DNA ligase 1 (LIG1) K126 in cells. The catalytic domain of G9a showed a higher affinity for di-methylated lysine on ATF7IP than LIG1, which may create different methylation levels of different substrates in cells. Furthermore, we found that M-phase phosphoprotein 8 (MPP8), known as a H3K9me3-binding protein, recognizes methylated ATF7IP via its chromodomain. MPP8 is also a known component of the human silencing hub complex that mediates silencing of transgenes via SETDB1 recruitment, which is a binding partner of ATF7IP. Although the interaction between ATF7IP and SETDB1 does not depend on ATF7IP methylation, we found that induction of SETDB1/MPP8-mediated reporter-provirus silencing is delayed in mESCs expressing only an un-methylatable mutant of ATF7IP.ConclusionsOur findings provide new insights into the roles of lysine methylation in non-histone substrates which are targeted by the G9a/GLP complex and suggest a potential function of ATF7IP methylation in SETDB1/MPP8-mediated transgene silencing.

Abstract 5636: Identification of bladder cancer-associated cancer-testis antigens-derived long peptides encompassing both CTL and promiscuous HLA class II-restricted Th cell epitopes

Abstract DEP domain containing 1 (DEPDC1) and M-phase phosphoprotein 1 (MPHOSPH1) are human cancer testis antigens that are frequently overexpressed in urinary bladder cancer. The growth of bladder cancer cells was significantly inhibited by knockdown of DEPDC1 or MPHOSPH1 expression using small interfering RNAs; thus, DEPDC1 and MPHOSPH1 are essential for tumor growth and survival, indicating the clinical significance of these antigens as targets for antigen-specific cancer immunotherapy. Subsequent reports further identified highly immunogenic DEPDC1- and MPHOSPH1-derived short peptides (SPs) that can induce HLA-A24-restricted cytotoxic T lymphocytes (CTLs) from peripheral blood mononuclear cells (PBMCs) isolated from patients with bladder cancer. These DEPDC1- and MPHOSPH1-derived SPs vaccine administered to patients with progressive bladder cancer was well tolerated and effectively activated SPs-specific CTLs in vivo, and the better CTL induction was associated with longer survival of patients in a recent phase I and II clinical trial (Obara W et al. Ann. Oncol. 28: 798, 2017). Here, we aimed to identify long peptides (LPs) derived from DEPDC1 and MPHOSPH1 that induced both T-helper (Th) cells and tumor-reactive CTLs. Stimulation of PBMCs isolated from healthy donors with the synthetic DEPDC1- and MPHOSPH1-LPs predicted to bind to promiscuous human HLA class II molecules by a computer algorithm induced specific CD4-positive T cells as revealed by interferon-gamma enzyme-linked immunospot assays. Three of six LPs encompassed SPs recognized by either HLA-A2- or -A24-restricted CTLs, and all six LPs stimulated DEPDC1- or MPHOSPH1-specific Th cells restricted by promiscuous and frequently observed HLA class II molecules in the Japanese population. Some LPs are naturally processed from the proteins in DCs, and the capacity of these LPs to cross-prime SP-specific CTLs was confirmed in vivo using HLA-A2 or -A24 transgenic mice. The LP-specific and HLA class II-restricted T-cell responses were also observed in PBMCs isolated from patients with bladder cancer. Repeated stimulation of PBMCs with DEPDC1-LPs and MPHOSPH1-LPs yielded clonal Th cells expressing specific T-cell receptor (TCR)-alpha and beta genes. These DEPDC1- or MPHOSPH1-derived LPs may have applications in immunotherapy in patients with bladder cancer, and the TCR genes identified may be useful for monitoring of Th cells specific to LPs in vivo. [ This research was supported by a MEXT Grant-in-Aid for Scientific Research on Innovative Areas 22133005; JSPS KAKENHI 23650609, 24300334, 15H04311 and 16H06498 for Scientific Research on Innovative Area "Neo-self"; the Project for Cancer Research And Therapeutic Evolution (P-CREATE) from the Japan Agency for Medical Research and Development, AMED; and OncoTherapy Science, Inc.. ] Citation Format: Yasuharu Nishimura, Miki Tsuruta, Shohei Ueda, Poh Yin Yew, Isao Fukuda, Sachiko Yoshimura, Hiroyuki Kishi, Hiroshi Hamana, Masatoshi Hirayama, Junji Yatsuda, Atsushi Irie, Satoru Senju, Eiji Yuba, Tomomi Kamba, Masatoshi Eto, Hideki Nakayama. Identification of bladder cancer-associated cancer-testis antigens-derived long peptides encompassing both CTL and promiscuous HLA class II-restricted Th cell epitopes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5636.

Knockdown of MPP8 suppresses cell proliferation via regulation of HOXA5 in non-small cell lung cancer cells.

M-phase phosphoprotein 8 (MPP8) is reported to be closely implicated in cancer initiation and progression. In addition, the homeobox gene HOXA5 has been shown to play critical roles in hematopoiesis, embryogenesis, and tumorigenesis. Nevertheless, the functional relevance of MPP8 and it's relation with HOXA5 in non-small cell lung cancer (NSCLC) is unknown. Therefore, the present study aimed to detect the expression profile of MPP8 in NSCLC and further explore it's biological roles in lung cancer cells. Cell proliferation was measured by CCK-8 assay and EdU incorporation assay. Real-time PCR was applied to detect the mRNA expression of MPP8 and HOXA5. The protein levels of MPP8 and HOXA5 were evaluated by western blot. Our study found that the expression of MPP8 was significantly increased in the NSCLC tissue compared with the adjacent non-tumorous tissue. Compared with the human lung fibroblasts, the elevated gene expression of MPP8 was also detected in the human NSCLC cell lines including NCI-H23 and NCI-H1299. In addition, knockdown of MPP8 led to an obvious reduction in cell viability and DNA synthesis in NCI-H23 and NCI-H1299 cells. Furthermore, down-regulation of MPP8 resulted in elevated expression of HOXA5 in NSCLC cells both at the mRNA and protein levels. Moreover, depletion of HOXA5 abolished the anti-tumor function of MPP8 knockdown in NSCLC cells. The present study demonstrated that MPP8 was associated with NSCLC cell proliferation through regulation of HOXA5, suggesting that MPP8 may act as a novel therapeutic target for treatment of NSCLC.