Overexpression of LIM and SH3 Protein 1 leading to accelerated G2/M phase transition contributes to enhanced tumourigenesis in oral cancer.

BACKGROUND: LIM and SH3 protein 1 (LASP-1) is a specific focal adhesion protein involved in several malignant tumors. However, its role in oral squamous cell carcinoma (OSCC) is unknown. The aim of this study was to characterize the role and molecular status/mechanism of LASP-1 in OSCC. METHODS: We evaluated LASP-1 mRNA and protein expressions in OSCC-derived cell lines and primary OSCCs. Using an shRNA system, we analyzed the effect of LASP-1 on the biology and function of the OSCC cell lines, HSC-3 and Ca9-22. The cells also were subcutaneously injected to evaluate tumor growth in vivo. Data were analyzed by the Fisher's exact test or the Mann-Whitney U test. Bonferroni correction was used for multiple testing. RESULTS: Significant up-regulation of LASP-1 was detected in OSCC-derived cell lines (n = 7, P<0.007) and primary OSCCs (n = 50, P<0.001) compared to normal controls. LASP-1 knockdown cells significantly inhibited cellular proliferation compared with shMock-transfected cells (P<0.025) by arresting cell-cycle progression at the G2 phase. We observed dramatic reduction in the growth of shLASP-1 OSCC xenografts compared with shMock xenografts in vivo. CONCLUSION: Our results suggested that overexpression of LASP-1 is linked closely to oral tumourigenicity and further provide novel evidence that LASP-1 plays an essential role in tumor cellular growth by mediating G2/M transition

Kinesin Family member 4A: A Potential Predictor for Progression of Human Oral Cancer

<div><p>Background</p><p>Kinesin family member 4A (KIF4A), a microtubule-based motor protein, was implicated in regulation of chromosomal structure and kinetochore microtubule dynamics. Considering the functions of KIF4A, we assumed that KIF4A is involved in progression of oral squamous cell carcinomas (OSCCs) via activation of the spindle assembly checkpoint (SAC). However, little is known about the relevance of KIF4A in the behavior of OSCC. We investigated the KIF4A expression status and its functional mechanisms in OSCC.</p> <p>Methods</p><p>The KIF4A expression levels in seven OSCC-derived cells were analyzed by quantitative reverse transcriptase-polymerase chain reaction and immunoblotting analyses. Using a KIF4A knockdown model, we assessed the expression of (SAC)-related molecules (BUB1, MAD2, CDC20, and cyclin B1), cell-cycle, and cellular proliferation. In addition to <i>in</i><i>vitro</i> data, the clinical correlation between the KIF4A expression levels in primary OSCCs (n = 106 patients) and the clinicopathologic status by immunohistochemistry (IHC) also were evaluated. </p> <p>Results</p><p>KIF4A mRNA and protein were up-regulated significantly (<i>P</i> < 0.05) in seven OSCC-derived cells compared with human normal oral keratinocytes. In the KIF4A knockdown cells, SAC activation was observed via increased BUB1 expression on the kinetochores, appropriate kinetochore localization of MAD2, down-regulation of CDC20, up-regulation of cyclin B1, and cell-cycle arrested at G2/M phase. The results showed that cellular proliferation of KIF4A knockdown cells decreased significantly (<i>P</i> < 0.05) compared with control cells. IHC showed that KIF4A expression in primary OSCCs was significantly (<i>P</i> < 0.05) greater than in the normal oral counterparts and that KIF4A-positive OSCCs were correlated closely (<i>P</i> < 0.05) with tumoral size. </p> <p>Conclusions</p><p>Our results proposed for the first time that KIF4A controls cellular proliferation via SAC activation. Therefore, KIF4A might be a key regulator for tumoral progression in OSCCs.</p> </div

Deciphering the Virome of Culex vishnui Subgroup Mosquitoes, the Major Vectors of Japanese Encephalitis, in Japan

Japanese encephalitis (JE) remains a public health concern in several countries, and the Culex mosquito plays a central role in its transmission cycle. Culex mosquitoes harbor a wide range of viruses, including insect-specific viruses (ISVs), and can transmit a variety of arthropod-borne viruses (arboviruses) that cause human and animal diseases. The current trend of studies displays enhanced efforts to characterize the mosquito virome through bulk RNA sequencing due to possible arbovirus&ndash;ISV interactions; however, the extent of viral diversity in the mosquito taxon is still poorly understood, particularly in some disease vectors. In this study, arboviral screening and RNA virome analysis of Culex tritaeniorhynchus and C. pseudovishnui, which are part of the Culex vishnui subgroup mosquitoes, were performed. Results from these two mosquito species, known as the major vectors of JE virus (JEV) in Asia, collected in three prefectures in Japan were also compared with the sympatric species C. inatomii. A total of 27 viruses, including JEV, were detected from these Culex mosquitoes. Molecular and phylogenetic analyses of the detected viruses classified 15 of the 27 viruses as novel species, notably belonging to the Flaviviridae, Rhabdoviridae, Totiviridae, and Iflaviridae families. The successful isolation of JEV genotype I confirmed its continuous presence in Japan, suggesting the need for periodic surveillance. Aside from JEV, this study has also reported the diversity of the RNA virome of disease vectors and broadened the knowledge on mosquito virome profiles containing both arbovirus and ISV. Mosquito taxon seemed to contribute largely to the virome structure (e.g., virome composition, diversity, and abundance) as opposed to the geographical location of the mosquito species. This study therefore offers notable insights into the ecology and evolution of each identified virus and viral family. To the authors&rsquo; knowledge, this is the first study to characterize the viromes of the major JE vectors in Japan

Evaluation of KIF4A protein expression in primary OSCCs.

<p>Representative IHC results for KIF4A protein in normal oral tissue (<b>A</b>) and primary OSCC (<b>B</b>). <b>A</b>, <b>B</b>: Original magnification, ×400. Scale bars, 10 μm. Strong KIF4A immunoreactivity was detected in primary OSCCs; normal oral tissues show almost negative immunostaining. <b>C</b>: The state of KIF4A protein expression in primary OSCCs (n=106) and the normal counterparts. The KIF4A IHC scores were calculated as follows: IHC score = 0× (number of unstained cells in the field) +1× (number of weakly stained cells in the field) +2× (number of moderately stained cells in the field) +3× (number of intensely stained cells in the field). The KIF4A IHC scores for normal oral tissues and OSCCs ranged from 26.5 to 103 (median, 66.5) and 62.3 to 188 (median, 131), respectively. KIF4A protein expression levels in OSCCs are significantly (<i>P</i> < 0.05, Mann-Whitney’s U test) higher than in normal oral tissues.</p

Expression of KIF4A in shKIF4A cells.

<p>(<b>A</b>) qRT-PCR shows that KIF4A mRNA expression in the shKIF4A cells (HSC-3-and Ca9-22-derived transfectants; 2 clones each) is significantly lower than in the shMock cells (<i>P</i> < 0.05, Mann-Whitney U test). (<b>B</b>) Immunoblotting analysis shows that the KIF4A protein levels in shKIF4A-transfected cells (HSC-3- and Ca9-22-derived transfectants; 2 clones each) also have decreased markedly compared with that in the shMock cells (<i>P</i> < 0.05, Mann-Whitney U test).</p

Localization of BUB1 and MAD2 during the prometaphase in shMock and shKIF4A cells.

<p>Localization of BUB1 and MAD2 to the kinetochores is compared in shKIF4A and shMock cells by immunofluorescence. (<b>A</b>) BUB1 on kinetochores increased in shKIF4A cells compared with that in the shMock cells (green, KIF4A; red, BUB1; blue, DNA). (<b>B</b>) Appropriate localization of MAD2 is seen in shKIF4A cells, whereas kinetochore localization is not seen in the shMock cells (green, KIF4A; red, MAD2; blue, DNA).</p

Reduced cellular growth in shKIF4A cells.

<p>To determine the effect of shKIF4A on cellular proliferation, shKIF4A and shMock cells were seeded in six-well plates at a density of 1×10⁴ viable cells/well. Both transfectants were counted on seven consecutive days. The cellular growth of shKIF4A-transfected cells (HSC-3- and Cas9-22-derived transfectants; 2 clones each) were significantly inhibited compared with shMock cells after 7 days (168 hours). The results were expressed as the means ± SEM of values from three assays. The asterisks indicate significant differences between the shKIF4A and shMock cells (<i>P</i> < 0.05, Mann-Whitney U test).</p

Evaluation of KIF4A expression in OSCC-derived cellular lines.

<p>(<b>A</b>) Quantification of KIF4A mRNA expression in OSCC-derived cellular lines by qRT-PCR analysis. Significant up-regulation of KIF4A mRNA is seen in seven OSCC-derived cellular lines compared with the HNOKs (<i>P</i> < 0.05, Mann-Whitney U test). Data are expressed as the means ± SEM of triplicate results. (<b>B</b>) Immunoblotting analysis of KIF4A protein in the OSCC-derived cellular lines and HNOKs. KIF4A protein expression is up-regulated in the OSCC-derived cellular lines compared with that in the HNOKs. Densitometric KIF4A protein data are normalized to GAPDH protein levels. The values are expressed as a percentage of the HNOKs (<i>P</i> < 0.05, Mann-Whitney U test).</p