AMPD3 is associated with the malignant characteristics of gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are mesenchymal tumors of the gastrointestinal tract. It is well known that activating mutations in the receptor tyrosine kinases KIT and platelet-derived growth factor receptor-α have essential roles in the pathogenesis of GISTs. The activation of these receptor protein kinases triggers multiple signaling pathways that promote cell proliferation and survival; however, the exact mechanism by which the activation of these kinases promotes the progression of GISTs remains uncertain. The aim of the present was to search for genes that are associated with the progression of GIST. The present study used reverse transcription-quantitative polymerase chain reaction to demonstrate that adenosine monophosphate deaminase 3 (AMPD3) was highly expressed in GISTs. Furthermore, transfection of GIST-T1 cells with KIT-specific small interfering RNA (siRNA) demonstrated that the expression of AMPD3 was dependent on KIT expression, while the depletion of AMPD3 in human GIST-T1 cells using AMPD3-specific siRNA resulted in the suppression of cell migration and invasion. In addition, AMPD3 depletion sensitized GIST-T1 cells to the tyrosine kinase inhibitor imatinib. The results of the present suggested that the combined inhibition of tyrosine kinases and AMPD3 may be effective for the treatment of GISTs.

Nek2 siRNA therapy using a portal venous port-catheter system for liver metastasis in pancreatic cancer.

Nek2 (NIMA-related kinase 2) is a serine-threonine kinase and human homolog of the mitotic regulator NIMA of Aspergillus nidulan. We reported the efficiency of Nek2 siRNA in several cancer xenograft models using cholangiocarcinoma, breast cancer and colorectal cancer. Pancreatic cancer is difficult to treat due to its rapid progression and resistance to chemotherapy. Novel treatments are urgently required to improve survival in pancreatic cancer, and siRNA are a promising therapeutic option. However, finding an in vivo drug delivery system of siRNA remains a major problem for clinical application. In this study, the overexpression of Nek2 was identified in pancreatic cancer cell lines. Nek2 siRNA inhibited tumor growth in a subcutaneous xenograft mouse model of pancreatic cancer, prolonged the survival time in an intraperitoneal xenograft mouse model and efficiently prevented the progression of liver metastasis using a portal venous port-catheter system. Taken together, Nek2 is an effective therapeutic target in pancreatic cancer. An adequate delivery system is considered important in treating advanced pancreatic cancer, such as peritoneal dissemination and liver metastasis. Further investigations are required on the safety and side effects of the portal venous port-catheter system. We hope that Nek2 siRNA will be a novel therapeutic strategy for pancreatic cancer with liver metastasis and peritoneal dissemination.

CAR T Cells Targeting Podoplanin Reduce Orthotopic Glioblastomas in Mouse Brains.

Glioblastoma (GBM) is the most common and lethal primary malignant brain tumor in adults with a 5-year overall survival rate of less than 10%. Podoplanin (PDPN) is a type I transmembrane mucin-like glycoprotein, expressed in the lymphatic endothelium. Several solid tumors overexpress PDPN, including the mesenchymal type of GBM, which has been reported to present the worst prognosis among GBM subtypes. Chimeric antigen receptor (CAR)-transduced T cells can recognize predefined tumor surface antigens independent of MHC restriction, which is often downregulated in gliomas. We constructed a lentiviral vector expressing a third-generation CAR comprising a PDPN-specific antibody (NZ-1-based single-chain variable fragment) with CD28, 4-1BB, and CD3ζ intracellular domains. CAR-transduced peripheral blood monocytes were immunologically evaluated by calcein-mediated cytotoxic assay, ELISA, tumor size, and overall survival. The generated CAR T cells were specific and effective against PDPN-positive GBM cells in vitro. Systemic injection of the CAR T cells into an immunodeficient mouse model inhibited the growth of intracranial glioma xenografts in vivo. CAR T-cell therapy that targets PDPN would be a promising adoptive immunotherapy to treat mesenchymal GBM.

UBE2S is associated with malignant characteristics of breast cancer cells.

Ubiquitination is essential for various biological processes, such as signal transduction, intracellular trafficking, and protein degradation. Accumulating evidence has demonstrated that ubiquitination plays a crucial role in cancer development. In this report, we examine the expression and function of ubiquitin-conjugating enzyme E2S (UBE2S) in breast cancer. Immunohistochemical analysis revealed that UBE2S is highly expressed in breast cancer. The depletion of UBE2S by siRNA induced disruption of the actin cytoskeleton and focal adhesions. Interestingly, phosphorylation of FAK at Tyr397, which is important for the transduction of integrin-mediated signaling, was significantly reduced by UBE2S knockdown. We also show that UBE2S knockdown suppressed the malignant characteristics of breast cancer cells, such as migration, invasion, and anchorage-independent growth. Our results indicate that UBE2S could be a potential target for breast cancer treatment.

FAM98A is a novel substrate of PRMT1 required for tumor cell migration, invasion, and colony formation.

Protein arginine methylation, which is mediated by a family of protein arginine methyltransferases (PRMTs), is associated with numerous fundamental cellular processes. Accumulating studies have revealed that the expression of multiple PRMTs promotes cancer progression. In this study, we examined the role of PRMT1 in ovarian cancer cells. PRMT1 is expressed in multiple ovarian cancer cells, and the depletion of its expression suppressed colony formation, in vivo proliferation, migration, and invasion. To gain insight into PRMT1-mediated cancer progression, we searched for novel substrates of PRMT1. We found that FAM98A, whose physiological function is unknown, was arginine-methylated by PRMT1. FAM98A is expressed in numerous ovarian cancer cell lines and is important for the malignant characteristics of ovarian cancer cells. Our results indicate the possible role of the PRMT1-FAM98A pathway in cancer progression.

TRIP13 is expressed in colorectal cancer and promotes cancer cell invasion.

Thyroid hormone receptor interactor 13 (TRIP13) is a member of the ATPases associated with various cellular activities family of proteins and is highly conserved in a wide range of species. Recent studies have demonstrated that TRIP13 is critical for the inactivation of the spindle assembly checkpoint and is associated with the progression of certain cancers. In the present study, the role of TRIP13 in colorectal cancer (CRC) was examined. Reverse transcription-quantitative polymerase chain reaction analysis revealed that TRIP13 messenger RNA was highly expressed in multiple CRC tissues. The depletion of TRIP13 in CRC cells suppressed cell proliferation, migration and invasion. To determine whether the catalytic activity of TRIP13 was critical for cancer progression, an inactive mutant of TRIP13 was expressed in CRC cells. The invasion of cancer cells that expressed the mutant TRIP13 was significantly reduced compared with that of the wild type TRIP13-expressing cancer cells. These results indicate that TRIP13 could be a potential target for CRC treatment.

Treatment with near-infrared radiation promotes apoptosis in pancreatic cancer cells.

Cancer remains one of the leading causes of human mortality worldwide. Radiation and chemotherapy are commonly used for cancer treatment; however, the combination of these therapies and surgery do not completely eradicate cancer cells. Near-infrared radiation (NIR) is a low-energy form of radiation that exerts multiple effects on mammalian cells. Previous studies have reported that NIR induces DNA double-strand breaks and apoptosis of cancer cells. In the present study, a 915-nm laser was used to examine the effects of NIR on pancreatic cancer cells. Irradiation of pancreatic cancer cells using a 915-nm laser significantly induced caspase-3 activation and apoptosis. In addition, the combination of gemcitabine treatment and a 915-nm laser synergistically increased the number of apoptotic cells. The results of the present study indicate the use of infrared irradiation and chemotherapy may be a possible therapy for the treatment of cancer.

HOXB13 and ALX4 induce SLUG expression for the promotion of EMT and cell invasion in ovarian cancer cells.

Homeoproteins, a family of transcription factors that have conserved homeobox domains, play critical roles in embryonic development in a wide range of species. Accumulating studies have revealed that homeoproteins are aberrantly expressed in multiple tumors and function as either tumor promoters or suppressors. In this study, we show that two homeoproteins, HOXB13 and ALX4, are associated with epithelial to mesenchymal transition (EMT) and invasion of ovarian cancer cells. HOXB13 and ALX4 formed a complex in cells, and exogenous expression of either protein promoted EMT and invasion. Conversely, depletion of either protein suppressed invasion and induced reversion of EMT. SLUG is a C2H2-type zinc-finger transcription factor that promotes EMT in various cell lines. Knockdown of HOXB13 or ALX4 suppressed SLUG expression, and exogenous expression of either protein promoted SLUG expression. Finally, we showed that SLUG expression was essential for the HOXB13- or ALX4-mediated EMT and invasion. Our results show that HOXB13/SLUG and ALX4/SLUG axes are novel pathways that promote EMT and invasion of ovarian cancer cells.

SATB2 suppresses the progression of colorectal cancer cells via inactivation of MEK5/ERK5 signaling.

Special AT-rich sequence binding protein 2 (SATB2) is an evolutionarily conserved transcription factor that has multiple roles in neuronal development, osteoblast differentiation, and craniofacial patterning. SATB2 binds to the nuclear matrix attachment region, and regulates the expression of diverse sets of genes by altering chromatin structure. Recent studies have reported that high expression of SATB2 is associated with favorable prognosis in colorectal and laryngeal cancer; however, it remains uncertain whether SATB2 has tumor-suppressive functions in cancer cells. In this study, we examined the effects of SATB2 expression on the malignant characteristics of colorectal cancer cells. Expression of SATB2 repressed the proliferation of cancer cells in vitro and in vivo, and also suppressed their migration and invasion. Extracellular signal-regulated kinase 5 (ERK5) is a mitogen-activated protein kinase that is associated with an aggressive phenotype in various types of cancer. SATB2 expression reduced the activity of ERK5, and constitutive activation of ERK5 restored the proliferation, anchorage-independent growth, migration and invasion of SATB2-expressing cells. Our results demonstrate the existence of a novel regulatory mechanism of SATB2-mediated tumor suppression via ERK5 inactivation.

Inhibition of SNW1 association with spliceosomal proteins promotes apoptosis in breast cancer cells.

RNA splicing is a fundamental process for protein synthesis. Recent studies have reported that drugs that inhibit splicing have cytotoxic effects on various tumor cell lines. In this report, we demonstrate that depletion of SNW1, a component of the spliceosome, induces apoptosis in breast cancer cells. Proteomics and biochemical analyses revealed that SNW1 directly associates with other spliceosome components, including EFTUD2 (Snu114) and SNRNP200 (Brr2). The SKIP region of SNW1 interacted with the N-terminus of EFTUD2 as well as two independent regions in the C-terminus of SNRNP200. Similar to SNW1 depletion, knockdown of EFTUD2 increased the numbers of apoptotic cells. Furthermore, we demonstrate that exogenous expression of either the SKIP region of SNW1 or the N-terminus region of EFTUD2 significantly promoted cellular apoptosis. Our results suggest that the inhibition of SNW1 or its associating proteins may be a novel therapeutic strategy for cancer treatment.

Paired related homeobox 1 is associated with the invasive properties of glioblastoma cells.

Glioblastoma is a highly proliferative and invasive tumor. Despite extensive efforts to develop treatments for glioblastoma, the currently available therapies have only limited effects. To develop novel strategies for glioblastoma treatment, it is crucial to elucidate the molecular mechanisms that promote the invasive properties of glioblastoma. In the present study, we showed that the paired related homeobox 1 (PRRX1) is associated with glioblastoma cell invasion. The depletion of PRRX1 suppressed the invasion and neurosphere formation of glioblastoma cells. Conversely, the exogenous expression of PRRX1 promoted invasion. The Notch signaling pathway, which is an evolutionarily conserved pathway that is essential for developmental processes, plays an important role in the tumorigenesis of glioblastoma. The expression of PRRX1 induced the activation of Notch signaling, and the inhibition of Notch signaling suppressed PRRX1-mediated cell invasion. Our results indicate that activation of Notch signaling by PRRX1 is associated with the promotion of glioblastoma cell invasion.

Special AT-rich sequence-binding protein 2 suppresses invadopodia formation in HCT116 cells via palladin inhibition.

Invadopodia are specialized actin-based microdomains of the plasma membrane that combine adhesive properties with matrix degrading activities. Proper functioning of the bone, immune, and vascular systems depend on these organelles, and their relevance in cancer cells is linked to tumor metastasis. The elucidation of the mechanisms driving invadopodia formation is a prerequisite to understanding their role and ultimately to controlling their functions. Special AT-rich sequence-binding protein 2 (SATB2) was reported to suppress tumor cell migration and metastasis. However, the mechanism of action of SATB2 is unknown. Here, we show that SATB2 inhibits invadopodia formation in HCT116 cells and that the molecular scaffold palladin is inhibited by exogenous expression of SATB2. To confirm this association, we elucidated the function of palladin in HCT116 using a knock down strategy. Palladin knock down reduced cell migration and invasion and inhibited invadopodia formation. This phenotype was confirmed by a rescue experiment. We then demonstrated that palladin expression in SATB2-expressing cells restored invasion and invadopodia formation. Our results showed that SATB2 action is mediated by palladin inhibition and the SATB2/palladin pathway is associated with invadopodia formation in colorectal cancer cells.

SHCBP1 is required for midbody organization and cytokinesis completion.

The centralspindlin complex, which is composed of MKLP1 and MgcRacGAP, is one of the crucial factors involved in cytokinesis initiation. Centralspindlin is localized at the middle of the central spindle during anaphase and then concentrates at the midbody to control abscission. A number of proteins that associate with centralspindlin have been identified. These associating factors regulate furrowing and abscission in coordination with centralspindlin. A recent study identified a novel centralspindlin partner, called Nessun Dorma, which is essential for germ cell cytokinesis in Drosophila melanogaster. SHCBP1 is a human ortholog of Nessun Dorma that associates with human centralspindlin. In this report, we analyzed the interaction of SHCBP1 with centralspindlin in detail and determined the regions that are required for the interaction. In addition, we demonstrate that the central region is necessary for the SHCBP1 dimerization. Both MgcRacGAP and MKLP1 are degraded once cells exit mitosis. Similarly, endogenous and exogenous SHCBP1 were degraded with mitosis progression. Interestingly, SHCBP1 expression was significantly reduced in the absence of centralspindlin, whereas centralspindlin expression was not affected by SHCBP1 knockdown. Finally, we demonstrate that SHCBP1 depletion promotes midbody structure disruption and inhibits abscission, a final stage of cytokinesis. Our study gives novel insight into the role of SHCBP in cytokinesis completion.

Silencing of STRN4 suppresses the malignant characteristics of cancer cells.

The striatin family of proteins, comprising STRN, STRN3 and STRN4, are multidomain-containing proteins that associate with additional proteins to form a large protein complex. We previously reported that STRN4 directly associated with protein kinases, such as MINK1, TNIK and MAP4K4, which are associated with tumor suppression or tumor progression. However, it remains unclear whether STRN4 is associated with tumor progression. In this report, we examined the role that STRN4 plays in cancer malignancy. We show that depletion of STRN4 suppresses proliferation, migration, invasion and the anchorage-independent growth of cancer cells. In addition, STRN4 knockdown increases the sensitivity of pancreatic cancer cells to gemcitabine. Finally, we show that STRN4 knockdown suppresses the proliferation and metastasis of cancer cells in mice. Our results demonstrate a possible role of STRN4 in tumor progression.

PLAGL2 regulates actin cytoskeletal architecture and cell migration.

Pleomorphic adenoma gene like-2 (PLAGL2), a member of the PLAG gene family, is a C2H2 zinc finger transcriptional factor that is involved in cellular transformation and apoptosis. In this report, we show that PLAGL2 is associated with the organization of stress fibers and with small guanosine triphosphatase (GTPase) activity. Depletion of PLAGL2 in two different ovarian cancer cell lines, ES-2 and HEY, induced activation of RhoA, whereas activity of Rac1 was suppressed. Organization of actin stress fibers and focal adhesions was significantly promoted by PLAGL2 knockdown in a RhoA-dependent manner. Conversely, exogenous expression of PLAGL2 in MDA-MB-231 cells, a breast cancer cell line, resulted in the activation of Rac1 and the inactivation of RhoA. In addition, PLAGL2 expression induced lamellipodia formation and disruption of stress fiber formation. Finally, we show that CHN1 expression is essential for Rac1 inactivation in PLAGL2-depleted cells. Our results demonstrate a crucial role of PLAGL2 in actin dynamics and give further insight into the role of PLAGL2 in cellular transformation and apoptosis.

Silencing of TBC1D15 promotes RhoA activation and membrane blebbing.

Membrane blebs are round-shaped dynamic membrane protrusions that occur under many physiological conditions. Membrane bleb production is primarily controlled by actin cytoskeletal rearrangements mediated by RhoA. Tre2-Bub2-Cdc16 (TBC) domain-containing proteins are negative regulators of the Rab family of small GTPases and contain a highly conserved TBC domain. In this report, we show that the expression of TBC1D15 is associated with the activity of RhoA and the production of membrane blebs. Depletion of TBC1D15 induced activation of RhoA and membrane blebbing, which was abolished by the addition of an inhibitor for RhoA signaling. In addition, we show that TBC1D15 is required for the accumulation of RhoA at the equatorial cortex for the ingression of the cytokinetic furrow during cytokinesis. Our results demonstrate a novel role for TBC1D15 in the regulation of RhoA during membrane blebbing and cytokinesis.

The role of PLK1-phosphorylated SVIL in myosin II activation and cytokinetic furrowing.

Polo-like kinase 1 (PLK1) is a widely conserved serine/threonine kinase that regulates progression of multiple stages of mitosis. Although extensive studies about PLK1 functions during cell division have been performed, it is still not known how PLK1 regulates myosin II activation at the equatorial cortex and ingression of the cleavage furrow. In this report, we show that an actin/myosin-II-binding protein, supervillin (SVIL), is a substrate of PLK1. PLK1 phosphorylates Ser238 of SVIL, which can promote the localization of SVIL to the central spindle and association with PRC1. Expression of a PLK1 phosphorylation site mutant, S238A-SVIL, inhibited myosin II activation at the equatorial cortex and induced aberrant furrowing. SVIL has both actin- and myosin-II-binding regions in the N-terminus. Expression of ΔMyo-SVIL (deleted of the myosin-II-binding region), but not of ΔAct-SVIL (deleted of actin-binding region), reduced myosin II activation and caused defects in furrowing. Our study indicates a possible role of phosphorylated SVIL as a molecular link between the central spindle and the contractile ring to coordinate the activation of myosin II for the ingression of the cleavage furrow.

ALX1 induces snail expression to promote epithelial-to-mesenchymal transition and invasion of ovarian cancer cells.

Ovarian cancer is a highly invasive and metastatic disease with a poor prognosis if diagnosed at an advanced stage, which is often the case. Recent studies argue that ovarian cancer cells that have undergone epithelial-to-mesenchymal transition (EMT) acquire aggressive malignant properties, but the relevant molecular mechanisms in this setting are not well-understood. Here, we report findings from an siRNA screen that identified the homeobox transcription factor ALX1 as a novel regulator of EMT. RNA interference-mediated attenuation of ALX1 expression restored E-cadherin expression and cell-cell junction formation in ovarian cancer cells, suppressing cell invasion, anchorage-independent growth, and tumor formation. Conversely, enforced expression of ALX1 in ovarian cancer cells or nontumorigenic epithelial cells induced EMT. We found that ALX1 upregulated expression of the key EMT regulator Snail (SNAI1) and that it mediated EMT activation and cell invasion by ALX1. Our results define the ALX1/Snail axis as a novel EMT pathway that mediates cancer invasion.