2-O-Octadecylascorbic acid represses RhoGDIß expression and ameliorates DNA damage-induced abnormal spindle orientations.

The appropriate regulation of spindle orientation maintains proper tissue homeostasis and avoids aberrant tissue repair or regeneration. Spindle misorientation due to imbalance or improper functioning leads to a loss of tissue integrity and aberrant growth, such as tissue loss or overgrowth. Pharmacological manipulation to prevent spindle misorientation will enable a better understanding of how spindle orientation is involved in physiological and pathological conditions and will provide therapeutic possibilities to treat patients associated with abnormal tissue function caused by spindle misorientation. N-terminal-deleted Rho guanine nucleotide dissociation inhibitor ß (RhoGDIß/RhoGDI2/LyGDI) produced by caspase-3 activation perturbs spindle orientation in surviving cells following exposure to either ionizing radiation or UVC. Thus, presumably, RhoGDIß cleaved by caspase-3 activation acts as a determinant of radiation-induced spindle misorientation that promote aberrant tissue repair due to deregulation of directional organization of cell population and therefore becomes a potential target of drugs to prevent such response. The objective of this study was to screen and identify chemicals that suppress RhoGDIß expression. We focused our attention on ascorbic acid (AA) derivatives because of their impact on the maintenance of skin tissue homeostasis. Here, we screened for AA derivatives that suppress RhoGDIß expression in HeLa cells and identified a lipophilic derivative, 2-O-octadecylascorbic acid (2-OctadecylAA), as a novel RhoGDIß inhibitor that ameliorated ionizing radiation-induced abnormal spindle orientations. Among all examined AA derivatives, which were also antioxidative, the inhibition activity was specific to 2-OctadecylAA. Therefore, this activity was not due to simple antioxidant properties. 2-OctadecylAA was previously shown to prevent hepatocellular carcinoma development. Our findings suggest that the anticarcinogenic effects of 2-OctadecylAA are partly due to RhoGDIß inhibition mechanisms by which spindle orientation perturbations are attenuated. Thus, the molecular targeting features of RhoGDIß warrant its further development for the treatment or control of spindle orientation abnormalities that affect epithelial homeostasis.

RhoGDIß affects HeLa cell spindle orientation following UVC irradiation.

The molecular signals that regulate mitotic spindle orientation to determine the proper division axis play a critical role in the development and maintenance of tissue homeostasis. However, deregulation of signaling events can result in spindle misorientation, which in turn can trigger developmental defects and cancer progression. Little is known about the cellular signaling pathway involved in the misorientation of proliferating cells that evade apoptosis after DNA damage. In this study, we found that perturbations to spindle orientation were induced in ultraviolet C (UVC)-irradiated surviving cells. N-terminal truncated Rho GDP-dissociation inhibitor ß (RhoGDIß), which is produced by UVC irradiation, distorted the spindle orientation of HeLa cells cultured on Matrigel. The short hairpin RNA-mediated knockdown of RhoGDIß significantly attenuated UVC-induced misorientation. Subsequent expression of wild-type RhoGDIß, but not a noncleavable mutant, RhoGDIß (D19A), again led to a relative increase in spindle misorientation in response to UVC. Our findings revealed that RhoGDIß impacts spindle orientation in response to DNA damage.

Inhibition of DMH-DSS-induced colorectal cancer by liposomal bovine lactoferrin in rats.

Bovine lactoferrin (bLF) is a multifunctional protein with anti-inflammatory, antibacterial, antiviral, anti-tumour and immunoregulatory effects. The present study was conducted to evaluate the anti-inflammatory and anti-tumour effects of liposomal bLF (LbLF) in a 1,2-dimethylhydrazine (DMH)/dextran sulphate sodium (DSS)-induced model of carcinogenesis in F344 rats. F344 rats were randomly divided into three groups: Control (water), 500 or 1,000 mg/kg/day LbLF; additionally, the rats were injected with DMH (20 mg/kg) once per week for 8 consecutive weeks, after one week of drinking water containing 1% DSS. All rats were sacrificed at 25 weeks. The tissues were examined for the presence of aberrant crypt foci (ACF) and subjected to histopathological analysis. Additionally, human colon cancer cells were utilised to investigate the effect of LbLF on proliferation and inflammation. Rats from the 500 and 1,000 mg/kg/day LbLF groups harboured significantly fewer colon ACF, adenomas and adenocarcinomas than the rats from the control group. Lastly, it was demonstrated that LbLF inhibits cell growth and TNF-α mRNA expression. These data support the hypothesis that LbLF affects colorectal carcinogenesis by suppressing inflammation and cell proliferation in rats.

Apoptosis­independent cleavage of RhoGDIß at Asp19 during PMA­stimulated differentiation of THP­1 cells to macrophages.

Rho GDP-dissociation inhibitor ß (RhoGDIß), a regulator of the Rho family of proteins, is expressed abundantly in the hematopoietic cell lineage. During apoptosis of hematopoietic cells, RhoGDIß is cleaved by caspase­3 at Asp19 and this cleaved form (Δ19­RhoGDIß) has been implicated in the apoptotic pathway. To clarify the role of RhoGDIß in hematopoietic cells, the present study performed immunoblotting and immunofluorescence staining to examine the expression of RhoGDIß and ∆19­RhoGDIß during phorbol 12­myristate 13­acetate (PMA)­stimulated differentiation of human THP­1 monocytic cells to macrophages. During differentiation of the THP­1 cells to macrophages, the expression of RhoGDIß remained stable; however, the expression of Δ19­RhoGDIß increased, particularly in well­spreading, non­apoptotic cells, which differentiated into macrophages. These results suggested that Δ19­RhoGDIß has an apoptosis­independent role in the PMA­induced differentiation of THP­1 cells to macrophages.

Janus face-like effects of Aurora B inhibition: antitumoral mode of action versus induction of aneuploid progeny.

The mitotic Aurora B kinase is overexpressed in tumors and various inhibitors for Aurora B are currently under clinical assessments. However, when considering Aurora B kinase inhibitors as anticancer drugs, their mode of action and the role of p53 status as a possible predictive factor for response still needs to be investigated. In this study, we analyzed the effects of selective Aurora B inhibition using AZD1152-HQPA/Barasertib (AZD1152) on HCT116 cells, U87-MG, corresponding isogenic p53-deficient cells and a primary glioblastoma cell line. AZD1152 treatment caused polyploidy and non-apoptotic cell death in all cell lines irrespective of p53 status and was accompanied by poly-merotelic kinetochore-microtubule attachments and DNA damage. In p53 wild-type cells a DNA damage response induced an inefficient pseudo-G1 cell cycle arrest, which was not able to halt ongoing endoreplication of cells. Of note, release of tumor cells from AZD1152 resulted in recovery of aneuploid progenies bearing numerical and structural chromosomal aberrations. Yet, AZD1152 treatment enhanced death receptor TRAIL-R2 levels in all tumor cell lines investigated. A concomitant increase of the activating natural killer (NK) cell ligand MIC A/B in p53-deficient cells and an induction of FAS/CD95 in cells containing p53 rendered AZD1152-treated cells more susceptible for NK-cell-mediated lysis. Our study mechanistically explains a p53-independent mode of action of a chemical Aurora B inhibitor and suggests a potential triggering of antitumoral immune responses, following polyploidization of tumor cells, which might constrain recovery of aneuploid tumor cells.

PARP6 acts as a tumor suppressor via downregulating Survivin expression in colorectal cancer.

Poly (ADP-ribose) polymerases (PARPs) are enzymes that transfer ADP-ribose groups to target proteins and are involved in a variety of biological processes. PARP6 is a novel member, and our previous findings suggest that PARP6 may act as a tumor suppressor via suppressing cell cycle progression. However, it is still unclear that PARP6 function besides growth suppression in colorectal cancer (CRC). In this study, we examined tumor suppressive roles of PAPR6 in CRC cells both in vitro and in vivo. We found that PARP6 inhibited colony formation, invasion and migration as well as cell proliferation. Moreover, ectopic overexpression of PARP6 decreased Survivin expression, which acts as an oncogene and is involved in apoptosis and mitosis. We confirmed the inverse correlation between PARP6 and Survivin expression in CRC cases by immunohistochemistry. Importantly, CRC cases with downregulation of PARP6 and upregulation of Survivin showed poor prognosis. In summary, PARP6 acts as a tumor suppressor via downregulating Survivin expression in CRC. PARP6 can be a novel diagnostic and therapeutic target together with Survivin for CRC.

Radiation-Induced RhoGDIß Cleavage Leads to Perturbation of Cell Polarity: A Possible Link to Cancer Spreading.

The equilibrium between proliferation and apoptosis is tightly balanced to maintain tissue homeostasis in normal tissues and even in tumors. Achieving and maintaining such a balance is important for cancer regrowth and spreading after cytotoxic treatments. Caspase-3 activation and tumor cell death following anticancer therapy as well as accompanying cell death pathways are well characterized, but their association to homeostasis of cancerous tissue and tumor progression remains poorly understood. Here we proposed a novel mechanism of cancer spreading induced by caspase-3. RhoGDIß, known as a direct cleavage substrate of caspase-3, is overexpressed in many epithelial cancers. The N-terminal-truncated RhoGDIß (ΔN-RhoGDIß) is accumulated in caspase-3-activated cells. Stable expression of ΔN-RhoGDIß in HeLa cells did not induce apoptosis, but impaired directional cell migration in a wound-healing assay accompanied by a perturbed direction of cell division at the wound edge. Subcellular protein fractionation experiments revealed that ΔN-RhoGDIß but not wild-type RhoGDIß was present in the detergent-soluble cytoplasmic and nuclear fractions and preferentially associated with Cdc42. Furthermore, Cdc42 activity was constitutively inhibited by stable expression of ΔN-RhoGDIß, resulting in increased radiation-induced compensatory proliferation linking to RhoA activation. Thus, ΔN-RhoGDIß dominant-negatively regulates Cdc42 activity and contributes to loss of polarity-related functions. The caspase-3-cleaved RhoGDIß is a possible determinant to promote cancer spreading due to deregulation of directional organization of tumor cell population and inhibition of default equilibrium between proliferation and apoptosis after cytotoxic damage. J. Cell. Physiol. 231: 2493-2505, 2016. © 2016 Wiley Periodicals, Inc.

tRNA modifying enzymes, NSUN2 and METTL1, determine sensitivity to 5-fluorouracil in HeLa cells.

Nonessential tRNA modifications by methyltransferases are evolutionarily conserved and have been reported to stabilize mature tRNA molecules and prevent rapid tRNA decay (RTD). The tRNA modifying enzymes, NSUN2 and METTL1, are mammalian orthologs of yeast Trm4 and Trm8, which are required for protecting tRNA against RTD. A simultaneous overexpression of NSUN2 and METTL1 is widely observed among human cancers suggesting that targeting of both proteins provides a novel powerful strategy for cancer chemotherapy. Here, we show that combined knockdown of NSUN2 and METTL1 in HeLa cells drastically potentiate sensitivity of cells to 5-fluorouracil (5-FU) whereas heat stress of cells revealed no effects. Since NSUN2 and METTL1 are phosphorylated by Aurora-B and Akt, respectively, and their tRNA modifying activities are suppressed by phosphorylation, overexpression of constitutively dephosphorylated forms of both methyltransferases is able to suppress 5-FU sensitivity. Thus, NSUN2 and METTL1 are implicated in 5-FU sensitivity in HeLa cells. Interfering with methylation of tRNAs might provide a promising rationale to improve 5-FU chemotherapy of cancer.

Survivin safeguards chromosome numbers and protects from aneuploidy independently from p53.

BACKGROUND: Survivin, a member of the inhibitor of apoptosis (IAP) gene family, has a dual role in mitosis and in apoptosis. It is abundantly expressed in every human tumor, compared with normal tissues. During mitosis Survivin assembles with the chromosomal passenger complex and regulates chromosomal segregation. Here, we aim to explore whether interference with the mitotic function of Survivin is linked to p53-mediated G1 cell cycle arrest and affects chromosomal stability. METHODS: In this study, we used HCT116, SBC-2, and U87-MG and generated corresponding isogenic p53-deficient cells. Retroviral vectors were used to stably knockdown Survivin. The resulting phenotype, in particular the mechanisms of cell cycle arrest and of initiation of aneuploidy, were investigated by Western Blot analysis, confocal laser scan microscopy, proliferation assays, spectral karyotyping and RNAi. RESULTS: In all cell lines Survivin-RNAi did not induce instant apoptosis but caused polyplodization irrespective of p53 status. Strikingly, polyploidization after knockdown of Survivin resulted in merotelic kinetochore spindle assemblies, γH2AX-foci, and DNA damage response (DDR), which was accompanied by a transient p53-mediated G1-arrest. That p53 wild type cells specifically arrest due to DNA damage was shown by simultaneous inhibition of ATM and DNA-PK, which abolished induction of p21waf/cip. Cytogenetic analysis revealed chromosomal aberrations indicative for DNA double strand break repair by the mechanism of non-homologous end joining (NHEJ), only in Survivin-depleted cells. CONCLUSION: Our findings suggest that Survivin plays an essential role in proper amphitelic kinetochore-spindle assembly and that constraining Survivin's mitotic function results in polyploidy and aneuploidy which cannot be controlled by p53. Therefore, Survivin critically safeguards chromosomal stability independently from p53.

The detergent-soluble cytoplasmic pool of survivin suppresses anoikis and its expression is associated with metastatic disease of human colon cancer.

Survivin is a component of the chromosomal passenger complex (CPC) that is essential for accurate chromosome segregation. Interfering with the function of Survivin in mitosis leads to chromosome segregation errors and defective cytokinesis. Survivin contains a Baculovirus IAP Repeat (BIR) and therefore was originally classified as inhibitor of apopotosis protein (IAP), yet its role in apoptosis after cellular stress remains largely unknown. We demonstrate here, that Survivin predominantly suppresses anoikis, a form of programmed cell death induced by loss of cellular adhesion to extracellular matrix. Interestingly, cells ectopically overexpressing EGFP-Survivin showed after loss of cell-matrix-interaction a decreased expression of IκB-α. Subsequent subcellular protein fractionation and immunoprecipitation experiments revealed that XIAP interacts with detergent-soluble Survivin which is known to cooperatively activate NF-κB signaling. Examination of the expression levels of detergent soluble Survivin in colorectal cancer cell lines and in colorectal cancerous tissues revealed that detergent soluble cytoplasmic Survivin levels correlated inversely with anoikis susceptibility in colorectal cancer. Therefore, the detergent soluble cytoplasmic Survivin might be a promising predictive biomarker for lymph node and distant metastases of colorectal cancer. We conclude that an anti-apoptotic function of detergent-soluble Survivin in interphase cells experiencing anoikis is mediated at least via XIAP/IκB-α/NF-κB signaling.

Centrosomal localization of RhoGDIß and its relevance to mitotic processes in cancer cells.

Rho GDP-dissociation inhibitors (RhoGDIs) are regulators of Rho family GTPases. RhoGDIß has been implicated in cancer progression, but its precise role remains unclear. We determined the subcellular localization of RhoGDIß and examined the effects of its overexpression and RNAi knockdown in cancer cells. Immunofluorescence staining showed that RhoGDIß localized to centrosomes in human cancer cells. In HeLa cells, exogenous GFP-tagged RhoGDIß localized to centrosomes and its overexpression caused prolonged mitosis and aberrant cytokinesis in which the cell shape was distorted. RNAi knockdown of RhoGDIß led to increased incidence of monopolar spindle mitosis resulting in polyploid cells. These results suggest that RhoGDIß has mitotic functions, including regulation of cytokinesis and bipolar spindle formation. The dysregulated expression of RhoGDIß may contribute to cancer progression by disrupting these processes.

PARP6, a mono(ADP-ribosyl) transferase and a negative regulator of cell proliferation, is involved in colorectal cancer development.

Poly(ADP-ribose) polymerase (PARP) is an enzyme that mediates post-translational modification of proteins. Seventeen known members of the PARP superfamily can be grouped into three classes based on catalytic activity: (i) classical poly(ADP-ribose) polymerases, (ii) mono(ADP­ribosyl) transferases and (iii) catalytically inactive members. PARP6 belongs to the mono(ADP-ribosyl) transferase class, and here we have found that PARP6 is a negative regulator of cell proliferation. Forced expression of PARP6 in HeLa cells induced growth suppression, but a PARP6 mutant with a C-terminal deletion lacking the catalytic domain had no effect. The PARP6-expressing cells accumulated in the S-phase, and the magnitude of S-phase accumulation was observed to be greater in cells expressing a PARP6 mutant with an N-terminal deletion, lacking a putative regulatory domain. Immunohistochemical analysis revealed that PARP6 positivity was found at higher frequencies in colorectal cancer tissues with well-differentiated histology compared to those with poorly differentiated histology. Furthermore, PARP6 positivity negatively correlated with the Ki-67 proliferation index. Kaplan-Meier analysis showed that PARP6-positive colorectal cancer had a good prognosis. Based on these results, we propose that PARP6 acts as a tumor suppressor through its role in cell cycle control.

Nuclear Aurora B and cytoplasmic Survivin expression is involved in lymph node metastasis of colorectal cancer.

The chromosomal passenger complex (CPC) is a key regulator of chromosome segregation and cytokinesis, and consists of Aurora B kinase, INCENP, Survivin and Borealin. Aurora B is a member of a family of serine/threonine protein kinases, and Survivin belongs to the inhibitors of apoptosis (IAP) gene family, and is also a member of the CPC family. Aurora B and Survivin have also been reported to be overexpressed in various human cancers; however, as yet no studies have investigated the co-expression of Survivin and Aurora B in colorectal carcinoma. Therefore, in the present study, the correlation between Aurora B and Survivin expression was investigated using immunohistochemistry and the associated pathological features in colorectal carcinoma were analyzed. Our present findings showed that nuclear Aurora B and cytoplasmic Survivin expression are strongly associated with and involved in lymph node metastasis in colorectal cancer. Therefore, we suggest that nuclear Aurora B and cytoplasmic Survivin are useful diagnostic markers and therapeutic targets in colorectal carcinoma.

Frequent increased gene copy number and high protein expression of tRNA (cytosine-5-)-methyltransferase (NSUN2) in human cancers.

NSUN2, also known as SAKI or MISU, is a methyltransferase which catalyses (cytosine-5-)-methylation of tRNA. The human NSUN2 gene is located on chromosome 5p15.31-33. We show that NSUN2 gene copy number is increased in oral and colorectal cancers. Protein expression levels of NSUN2 were determined by immunoblot using novel polyclonal antibodies raised against a synthetic peptide corresponding to the C-terminal region of the protein. In most normal tissues, NSUN2 expression levels were extremely low. On the other hand, oral and colorectal cancers typically expressed high levels of NSUN2. The level of NSUN2 was similar in interphase and mitotic cells, and immunohistochemical analysis demonstrated strong staining for NSUN2 in oral and colon cancer tissues when compared with normal tissues, providing a distinct diagnostic significance for NSUN2 in comparison with Ki-67, a widely used marker of actively proliferating cells. In addition, elevated protein expression of NSUN2 was confirmed by immunohistochemical analysis of various cancers including esophageal, stomach, liver, pancreas, uterine cervix, prostate, kidney, bladder, thyroid, and breast cancers. NSUN2 is regulated by Aurora-B, a newly developed molecular target for cancer therapy, leading us to propose that NSUN2 might become a valuable target for cancer therapy and a cancer diagnostic marker.

Giant cell glioblastoma is associated with altered aurora b expression and concomitant p53 mutation.

Giant cell glioblastoma (gcGB), a subtype of GB, is characterized by the presence of numerous multinucleated giant cells. The prognosis for gcGB is poor, but it may have a better clinical outcome compared with classic GB. The molecular alterations that lead to the multinucleated cell phenotype of gcGB have not been elucidated. Giant cell GB has a higher frequency of the tumor suppressor protein p53 mutations than GB, however, and a role for the mitotic Aurora B kinase has been suggested. We analyzed Aurora B expression in gcGB (n = 28) and GB (n = 54) patient tumor samples by immunohistochemistry; 17 gcGB and 22 GB samples were analyzed at the DNA and mRNA levels. No mutations in the Aurora B gene (AURKB) were found, but its mRNA and protein levels were significantly higher in gcGB than in GB. Fifty-nine percent of gcGB samples but only 18% of the GB samples showed p53 mutations. Ectopic overexpression of Aurora B induced a significant increase inthe proportion of multinucleated cells in p53 mutant U373-MG, but not in p53 wild-type U87-MG, glioma cells. RNAi of p53 in U87-MG cells led to an increase in the fraction of multinucleated cells that was further augmented by ectopic overexpression of Aurora B. These results suggest that loss of p53 function and dysregulated Aurora B protein levels might represent factors that drive the development of multinucleated cells in gcGB.

Differential regulation of caspase-9 by ionizing radiation- and UV-induced apoptotic pathways in thymic cells.

In mouse thymic lymphoma 3SB cells bearing wild type p53, ionizing radiation (IR) and UV light are potent triggers of caspase-3-dependent apoptosis. Although cytochrome c was released from mitochondria as expected, caspase-9 activation was not observed in UV-exposed cells. Laser scanning confocal microscopy analysis showed that caspase-9 is localized in an unusual punctuated pattern in UV-induced apoptotic cells. In agreement with differences in the status of caspase-9 activation between IR and UV, subcellular protein fractionation experiments showed that pro-apoptotic apoptosis protease-activating factor 1 (Apaf-1), normally a part of the apoptosome assembled in response to the release of cytochrome c from mitochondria, and B-cell lymphoma extra long (Bcl-xL), an inhibitor of the change in mitochondrial membrane permeability, were redistributed by the IR-exposure but not by the UV-exposure. Instead of the sequestration of the capase-9/apoptosome activation in UV-induced apoptotic cells, the extrinsic apoptotic signaling generated by caspase-8 activation and consequent activation of B-cell lymphoma extra long (Bid) to release cytochrome c from mitochondria was observed. Thus, the post-mitochondrial apoptotic pathway downstream of cytochrome c release cannot operate the apoptosome function in UV-induced apoptosis in thymic 3SB cells. The intracellular redistribution and sequestration of apoptosis-related proteins upon mitochondrion-based apoptotic signaling was identified as a novel cellular mechanism to respond to DNA damage in an agent type-specific manner. This finding suggests that the kind of the critical ultimate apoptosis-inducing DNA lesion complex form resulting from the agent-specific DNA damage responses is important to determine which of apoptosis signals would be activated.

Oncogenic role of nuclear accumulated Aurora-A.

Aurora-A, also known as Aik, BTAK, or STK15, is a centrosomal serine/threonine protein kinase, which is proto-oncogenic and is overexpressed in a wide range of human cancers. Besides gene amplification and mRNA overexpression, proteolytic resistance mechanisms are thought to contribute to overexpression of Aurora-A. However, it is not yet clear how overexpressed Aurora-A affects the expression of transformed phenotype. Here, we found that nuclear accumulation of Aurora-A was critical for transformation activity. Cellular protein fractionation experiments and immunoblot analysis demonstrated a predominance of Aurora-A in the nuclear soluble fraction in head and neck cancer cells. Indirect immunofluorescence using confocal laser microscopy confirmed nuclear Aurora-A in head and neck cancer cells, while most oral keratinocytes exhibited only centrosomal localization. The expression of nuclear export signal-fused Aurora-A demonstrated that the oncogenic transformation activity was lost on disruption of the nuclear localization. Thus, the cytoplasmic localization of overexpressed Aurora-A previously demonstrated by immunohistochemical analysis is not likely to correspond to that in intact cancer cells. This study identifies an alternative mode of Aurora-A overexpression in cancer, through nuclear rather than cytoplasmic functions. We suggest that substrates of Aurora-A in the cell nuclear soluble fraction can represent a novel therapeutic target for cancer.

Constitutive phosphorylation of aurora-a on ser51 induces its stabilization and consequent overexpression in cancer.

BACKGROUND: The serine/threonine kinase Aurora-A (Aur-A) is a proto-oncoprotein overexpressed in a wide range of human cancers. Overexpression of Aur-A is thought to be caused by gene amplification or mRNA overexpression. However, recent evidence revealed that the discrepancies between amplification of Aur-A and overexpression rates of Aur-A mRNA were observed in breast cancer, gastric cancer, hepatocellular carcinoma, and ovarian cancer. We found that aggressive head and neck cancers exhibited overexpression and stabilization of Aur-A protein without gene amplification or mRNA overexpression. Here we tested the hypothesis that aberration of the protein destruction system induces accumulation and consequently overexpression of Aur-A in cancer. PRINCIPAL FINDINGS: Aur-A protein was ubiquitinylated by APC(Cdh1) and consequently degraded when cells exited mitosis, and phosphorylation of Aur-A on Ser51 was observed during mitosis. Phosphorylation of Aur-A on Ser51 inhibited its APC(Cdh1)-mediated ubiquitylation and consequent degradation. Interestingly, constitutive phosphorylation on Ser51 was observed in head and neck cancer cells with protein overexpression and stabilization. Indeed, phosphorylation on Ser51 was observed in head and neck cancer tissues with Aur-A protein overexpression. Moreover, an Aur-A Ser51 phospho-mimetic mutant displayed stabilization of protein during cell cycle progression and enhanced ability to cell transformation. CONCLUSIONS/SIGNIFICANCE: Broadly, this study identifies a new mode of Aur-A overexpression in cancer through phosphorylation-dependent inhibition of its proteolysis in addition to gene amplification and mRNA overexpression. We suggest that the inhibition of Aur-A phosphorylation can represent a novel way to decrease Aur-A levels in cancer therapy.

Aurora-B expression and its correlation with cell proliferation and metastasis in oral cancer.

Aurora-B kinase is a chromosomal passenger protein and is essential for chromosome segregation and cytokinesis. Aurora-B overexpression in various cancer cells induces chromosomal number instability to produce multinuclearity and relates to metastasis. Here, we examined the expression of Aurora-B in oral squamous cell carcinoma (OSCC) to elucidate the relationship between Aurora-B expression and clinico-pathological findings by immunohistochemistry. Aurora-B expression was observed in normal oral squamous epithelia and OSCC cases, but the number of positive cells was significantly higher in OSCC than in normal squamous epithelium (p < 0.01). The labeling index of Aurora-B was significantly correlated with lymph node metastasis (p < 0.01) and histological grades of differentiation (p < 0.01). We also compared Aurora-B expression with Ki-67 expression and a positive correlation was found (p < 0.0001). Moreover, Aurora-B expression is significantly more frequent in multinuclear tumor cells than in total tumor cells. In summary, we found that Aurora-B expression was well correlated with cell proliferation, induction of multinuclear cells, histological differentiation, and metastasis in OSCC. These findings suggest that Aurora-B may be involved in tumor progression and that Aurora-B can be a new diagnostic and therapeutic target for OSCC.

Activation of Rac1 by Rho-guanine nucleotide dissociation inhibitor-beta with defective isoprenyl-binding pocket.

Rho-guanine nucleotide dissociation inhibitor-beta (RhoGDIbeta), a regulator for Rho GTPases, is implicated in cancer cell progression. We reported that C-terminal truncated RhoGDIbeta (DeltaC(166-201)-RhoGDIbeta) promoted metastasis through activating Rac1 signaling pathway in ras-transformed fibroblast cells. To better understand the mechanism of Rac1 activation by DeltaC(166-201)-RhoGDIbeta during metastasis, the amount of GTP-bound Rac1 was measured as the activation level of Rac1 in cells expressing various mutant RhoGDIbeta with sequential C-terminal deletions. Three C-terminal hydrophobic amino acid residues (Trp191, Leu193, and Ile195) supposed to interact with isoprenyl groups of Rac1, was indispensable for a proper regulation of Rac1 activation/inhibition. Deletion of this region led RhoGDIbeta to continuously associate with GTP-bound Rac1, provoking constitutive activation of Rac1. Thus, impaired interaction of RhoGDIbeta with Rac1 isoprenyl groups possibly makes RhoGDIbeta function as a positive regulator for Rac1 during metastasis.