Yoichiro Kajita

Mesenchymal stem cells cultured under hypoxia escape from senescence via down-regulation of p16 and extracellular signal regulated kinase.

Hypoxia has been considered to affect the properties of tissue stem cells including mesenchymal stem cells (MSCs). Effects of long periods of exposure to hypoxia on human MSCs, however, have not been clearly demonstrated. MSCs cultured under normoxic conditions (20% pO(2)) ceased to proliferate after 15-25 population doublings, while MSCs cultured under hypoxic conditions (1% pO(2)) retained the ability to proliferate with an additional 8-20 population doublings. Most of the MSCs cultured under normoxic conditions were in a senescent state after 100days, while few senescent cells were found in the hypoxic culture, which was associated with a down-regulation of p16 gene expression. MSCs cultured for 100days under hypoxic conditions were superior to those cultured under normoxic conditions in the ability to differentiate into the chondro- and adipogenic, but not osteogenic, lineage. Among the molecules related to mitogen-activated protein kinase (MAPK) signaling pathways, extracellular signal regulated kinase (ERK) was significantly down-regulated by hypoxia, which helped to inhibit the up-regulation of p16 gene expression. Therefore, the hypoxic culture retained MSCs in an undifferentiated and senescence-free state through the down-regulation of p16 and ERK.

Development of a Multiplex RNA Urine Test for the Detection and Stratification of Transitional Cell Carcinoma of the Bladder

Purpose: New markers that enable the percentage of transitional cell carcinomas (TCC) of the bladder that are diagnosed before invasion of the bladder muscle layers to be increased would reduce the morbidity and mortality associated with this disease. The purpose of this study was to develop a simple, accurate urine test based on mRNA markers and simple gene signatures that (a) could detect TCC before muscle invasion while maintaining high specificity in patients with hematuria or urinary tract infections and (b) identify patients most likely to have grade 3 or stage ≥T1 disease. Experimental Design: RNA markers with high overexpression in stage Ta tumors and/or T1 to T4 tumors but low expression in blood or inflammatory cells were characterized by quantitative reverse transcription-PCR using 2 mL of voided urine from 75 TCC patients and 77 control patients with other urological diseases. Results: A combination of the RNAs CDC2, MDK, IGFBP5, and HOXA13 detected 48%, 90%, and 100% of stage Ta, T1, and >T1 TCCs, respectively, at a specificity of 85%. Detection of Ta tumors increased to 60% for primary (non-recurrent) Ta tumors and 76% for Ta tumors ≥1 cm in diameter. Test specificity was 80% for the 20 control patients with urinary tract infections. The combination of CDC2 and HOXA13 distinguished between grade 1 to 2 TCCs and grade 3 or stage ≥T1 TCCs with ∼80% specificity and sensitivity. Conclusions: Simple gene expression signatures can be used as urine markers for the accurate detection and characterization of bladder cancer.

Activation of Rac1 Is Closely Related to Androgen-Independent Cell Proliferation of Prostate Cancer Cells Both in Vitro and in Vivo

We and others previously showed that signaling through cSrc or atypical protein kinase C (aPKC) pathway regulates the proliferation of prostate cancer cells and is associated with their progression to castrate-resistance in vivo. However, the interrelation of these two kinases has been largely unexplored. In the present study, we show that androgen-induced activation of cSrc regulates the activity of aPKC through the small molecular weight G protein Rac1 in androgen-dependent LNCaP cells. Knockdown of cSrc in those cells reduces the phosphorylation of aPKC and the abundance of activated form of Rac1. Additionally, the treatment of those cells with Rac1 inhibitor repressed cell cycle progression at G(1)/S transition. In fact, forced expression of a constitutively active Rac1 mutant in LNCaP cells promoted cell proliferation under androgen-depleted conditions both in vitro and in vivo. Moreover, LNCaP C4-2 and AILNCaP cells, the syngeneic androgen-independent sublines from LNCaP cells, harbored abundant Rac1-GTP. Importantly, the inhibition of Rac1 suppressed cell proliferation and induced apoptotic cell death in all prostate cancer cell lines tested irrespective of their androgen-dependence. In immunohistochemical evaluation of tumor specimens from prostate cancer patients, Rac1 pathway appeared to be activated in the majority of castrate-resistant diseases. Collectively, our present results both in vitro and in vivo highly implicate that Rac1 can be a potential therapeutic target for patients with advanced prostate cancer, especially those with castrate-resistant status.

A novel tumor-related protein, c7orf24, identified by proteome differential display of bladder urothelial carcinoma

Proteome analysis of bladder cancer with narrow‐range pH 2‐DE has identified a novel protein on chromosome 7 encoded by ORF 24 (C7orf24) as one of the highly expressed proteins in cancer cells. C7orf24 is currently registered in the protein database as a hypothetical protein with unknown function. The homologs of C7orf24 in other animals have also been registered as putative protein genes. Western blot analysis using a mAb against C7orf24 confirmed its higher expression in bladder cancer compared with normal tissue. Several other cancer cell lines were also found to express C7orf24. However, the introduction of C7orf24 into Rat‐1 or NIH3T3 cells did not cause malignant transformation. A stable transfectant of NIH3T3 cells with recombinant retrovirus vector was produced for a growth rate assay, and a higher growth rate was observed in C7orf24‐expressing cells compared with the controls. Six kinds of small interfering RNAs (siRNAs) were then produced, and C7orf24‐siRNA#5 showed a strong knockdown effect on protein expression and significant antiproliferative effects on cancer cell lines were demonstrated by the MTT assay. Therefore, C7orf24 may have an important role in cancer cell proliferation, and may be an appropriate therapeutic target molecule against cancer.

SPA-1 controls the invasion and metastasis of human prostate cancer.

Recent studies suggest that SIPA1 encoding a Rap GTPase‐activating protein SPA‐1 is a candidate metastasis efficiency‐modifying gene in human breast cancer. In this study, we investigated the expression and function of SPA‐1 in human prostate cancer (CaP). Immunohistochemical studies of tumor specimens from CaP patients revealed a positive correlation of SPA‐1 expression with disease progression and metastasis. The correlation was recapitulated in human CaP cell lines; LNCaP that rarely showed metastasis in SCID mice expressed an undetectable level of SPA‐1, whereas highly metastatic PC3 showed abundant SPA‐1 expression. Moreover, SIPA1 transduction in LNCaP caused prominent abdominal lymph node metastasis without affecting primary tumor size, whereas shRNA‐mediated SIPA1 knockdown or expression of a dominant‐active Rap1 mutant (Rap1V12) in PC3 suppressed metastasis. LNCaP transduced with SPA‐1 (LNCaP/SPA‐1) showed attenuated adhesion to the precoated extracellular matrices (ECM) including collagens and fibronectin, due to defective ECM‐medicated Rap1 activation. In addition, LNCaP/SPA‐1 showed a diminished level of nuclear Brd4, which is known to bind SPA‐1, resulting in reduced expression of a series of ECM‐related genes. These results suggest that SPA‐1 plays an important role in controlling metastasis efficiency of human CaP by regulating the expression of and interaction with ECM in the primary sites. (Cancer Sci 2011; 102: 828–836)

Dicoumarol enhances doxorubicin-induced cytotoxicity in p53 wild-type urothelial cancer cells through p38 activation

To investigate the effectiveness of a combined treatment of 3–30‐methylene‐bis[4‐hydroxycoumarin] (dicoumarol) with doxorubicin for the treatment of urothelial cancer, as doxorubicin is a common chemotherapeutic agent but its therapeutic efficacy is limited.

The Transcription Factor Sp3 Regulates the Expression of a Metastasis-Related Marker of Sarcoma, Actin Filament-Associated Protein 1-Like 1 (AFAP1L1)

We previously identified actin filament-associated protein 1-like 1 (AFAP1L1) as a metastasis-predicting marker from the gene-expression profiles of 65 spindle cell sarcomas, and demonstrated the up-regulation of AFAP1L1 expression to be an independent risk factor for distant metastasis in multivariate analyses. Little is known, however, about how the expression of AFAP1L1 is regulated. Luciferase reporter assays showed tandem binding motives of a specificity protein (Sp) located at −85 to −75 relative to the transcriptional start site to be essential to the promoter activity. Overexpression of Sp1 and Sp3 proteins transactivated the proximal AFAP1L1 promoter construct, and electrophoretic mobility shift assays showed that both Sp1 and Sp3 were able to bind to this region in vitro. Chromatin immunoprecipitation experiments, however, revealed that Sp3 is the major factor binding to the proximal promoter region of the AFAP1L1 gene in AFAP1L1- positive cells. Treatment with mithramycin A, an inhibitor of proteins binding to GC-rich regions, prevented Sp3 from binding to the proximal promoter region of AFAP1L1 and decreased its expression in a dose-dependent manner. Finally, knocking down Sp3 using small inhibitory RNA duplex (siRNA) reduced AFAP1L1 expression significantly, which was partially restored by expressing siRNA-resistant Sp3. These findings indicate a novel role for Sp3 in sarcomas as a driver for expression of the metastasis-related gene AFAP1L1.

Identification of AFAP1L1 as a prognostic marker for spindle cell sarcomas

Spindle cell sarcomas consist of tumors with different biological features, of which distant metastasis is the most ominous sign for a poor prognosis. However, metastasis is difficult to predict on the basis of current histopathological analyses. We have identified actin filament-associated protein 1-like 1 (AFAP1L1) as a candidate for a metastasis-predicting marker from the gene expression profiles of 65 spindle cell sarcomas. A multivariate analysis determined that AFAP1L1 was an independent factor for predicting the occurrence of distant metastasis (P=0.0001), which was further confirmed in another set of 41 tumors by a quantitative mRNA expression analysis. Immunohistochemical staining using paraffin-embedded tumor tissues revealed that the metastasis-free rate was significantly better in tumors negative for AFAP1L1 (P=0.0093 by log-rank test). Knocking down the AFAP1L1 gene in sarcoma cells resulted in inhibition of the cell invasion, and forced expression of AFAP1L1 in immortalized human mesenchymal stem cells induced anchorage-independent growth and increased cell invasiveness with high activity levels of matrix metallopeptidase. Furthermore, tumor growth in vivo was accelerated in AFAP1L1-transduced sarcoma cell lines. These results suggest that AFAP1L1 has a role in the progression of spindle cell sarcomas and is a prognostic biomarker.

AFAP1L1, a novel associating partner with vinculin, modulates cellular morphology and motility, and promotes the progression of colorectal cancers

We have previously identified actin filament‐associated protein 1‐like 1 (AFAP1L1) as a metastasis‐predicting marker for spindle cell sarcomas by gene expression profiling, and demonstrated that AFAP1L1 is involved in the cell invasion process by in vitro analyses. However, its precise molecular function has not been fully elucidated, and it remains unknown whether AFAP1L1 could be a prognostic marker and/or therapeutic target of other malignancies. In this study, we found a marked elevation of AFAP1L1 gene expression in colorectal cancer (CRC) tissues as compared to the adjacent normal mucosa. Multivariate analysis revealed that AFAP1L1 was an independent and significant factor for the recurrence of rectal cancers. Moreover, the addition of the AFAP1L1 expression level to the lymph node metastasis status provided more predictive information regarding postoperative recurrence in rectal cancers. AFAP1L1‐transduced CRC cells exhibited a rounded shape, increased cell motility on planar substrates, and resistance to anoikis in vitro. AFAP1L1 localized to the ringed structure of the invadopodia, together with vinculin, and AFAP1L1 was identified as a novel associating partner of vinculin by immunoprecipitation assay. AFAP1L1‐transduced cells showed accelerated tumor growth in vivo, presumably reflecting the anoikis resistance of these AFAP1L1‐expressing cells. Furthermore, the local administration of a siRNA against AFAP1L1 significantly suppressed the in vivo tumor growth of xenografts, suggesting that AFAP1L1 might be a candidate therapeutic target for CRCs. These results suggest that AFAP1L1 plays a role in the progression of CRCs by modulating cell shape and motility and by inhibiting anoikis, presumably through interactions with vinculin‐including protein complexes.