Toshinari Yamasaki

JunB promotes cell invasion and angiogenesis in VHL -defective renal cell carcinoma

Inactivation of the von Hippel–Lindau (VHL) tumor-suppressor gene causes both hereditary and sporadic clear-cell renal-cell carcinoma (ccRCC). Although the best-characterized function of the VHL protein (pVHL) is regulation of hypoxia-inducible factor-α (HIFα), pVHL also controls the development of pheochromocytoma through HIF-independent pathways by regulating JunB. However, it is largely unknown how these pathways contribute to the development and progression of ccRCC. In the present study, we confirmed that JunB was upregulated in VHL-defective ccRCC specimens by immunostaining. Short-hairpin RNA (shRNA)-mediated knockdown of JunB in 786-O and A498 VHL null ccRCC cells suppressed their invasiveness. In addition, JunB knockdown significantly repressed tumor growth and microvessel density in xenograft tumor assays. Conversely, forced expression of wild-type, but not dimerization-defective, JunB in a VHL-restored 786-O subclone promoted invasion in vitro and tumor growth and vessel formation in vivo. Quantitative PCR array analysis revealed that JunB regulated multiple genes relating to tumor invasion and angiogenesis such as matrix metalloproteinase-2 (MMP-2), MMP-9 and chemokine (C-C motif) ligand-2 (CCL2) in 786-O cells. JunB knockdown in these cells reduced the proteolytic activity of both MMPs in gelatin zymography and the amount of CCL2 in the culture supernatant. Moreover, shRNA-mediated knockdown of MMP-2 or inhibition of CCL2 activity with a neutralizing antibody repressed xenograft tumor growth and angiogenesis. Collectively, these results suggest that JunB promotes tumor invasiveness and enhances angiogenesis in VHL-defective ccRCCs.

The effect of ABCG2 genotype on the population pharmacokinetics of sunitinib in patients with renal cell carcinoma.

Background: Sunitinib, a multitargeted tyrosine kinase inhibitor, offers favorable therapeutic outcomes to patients with advanced renal cell carcinoma. However, to maximize the clinical benefits, an effective therapeutic management strategy with dose optimization is essential. The objectives of this analysis were to describe the pharmacokinetics (PK) of sunitinib by a population PK approach and to quantitatively evaluate the effect of potential predictive factors including ABCG2 genotype on the PK of sunitinib. Methods: Plasma concentration–time profiles at 3 consecutive days including a total of 245 sunitinib plasma concentrations were available from 19 Japanese patients with renal cell carcinoma. Blood samples were collected on days 2, 8, and 15 after the start of the therapy. Population PK analysis was performed using NONMEM 7.2. Body weight, gender, and genotype of ABCG2 421C>A were evaluated as potential covariates. Interoccasion variability (IOV) among the 3 sampling days was also assessed as a random effect parameter. Results: The sunitinib PK profiles were best described by a 1-compartment model with first-order absorption. The ABCG2 421C>A genotype was identified as a significant covariate for the prediction of oral clearance (CL/F). No significant improvement in model fit was observed by including body weight and/or gender. A systematic difference in estimated population CL/F was observed between days 2 and 8, which was quantified as approximately 30% decrease over time. This difference was described as a covariate for CL/F in the model. IOV included as a random effect parameter significantly improved the model fit. Conclusions: This analysis provides a population PK model of sunitinib with the ABCG2 421C>A genotype as a predictive covariate for CL/F. It also suggests that IOV and change of CL/F over time need to be considered to predict the sunitinib PK more accurately. These findings will be implemented to optimize the pharmacotherapy of sunitinib.

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.

Basic fibroblast growth factor causes urinary bladder overactivity through gap junction generation in the smooth muscle

Overactive bladder is a highly prevalent clinical condition that is often caused by bladder outlet obstruction (BOO). Increased coupling of bladder smooth muscle cells (BSMC) via gap junctions has been hypothesized as a mechanism for myogenic bladder overactivity in BOO, although little is known about the regulatory system underlying such changes. Here, we report the involvement of basic fibroblast growth factor (bFGF) and connexin 43, a bladder gap junction protein, in bladder overactivity. BOO created by urethral constriction in rats resulted in elevated bFGF and connexin 43 levels in the bladder urothelium and muscle layer, respectively, and muscle strips from these bladders were more sensitive than those from sham-operated controls to a cholinergic agonist. In vitro bFGF treatment increased connexin 43 expression in cultured rat BSMC via the ERK 1/2 pathway. This finding was supported by another in vivo model, where bFGF released from gelatin hydrogels fixed on rat bladder walls caused connexin 43 upregulation and gap junction formation in the muscle layer. Bladder muscle strips in this model showed increased sensitivity to a cholinergic agonist that was blocked by inhibition of gap junction function with alpha-glycyrrhetinic acid. Cystometric analyses of this model showed typical features of detrusor overactivity such as significantly increased micturition frequency and decreased bladder capacity. These findings suggest that bFGF from the urothelium could induce bladder hypersensitivity to acetylcholine via gap junction generation in the smooth muscle, thereby contributing to the myogenic overactivity of obstructed bladders.

Genome-wide analysis identifies a tumor suppressor role for aminoacylase 1 in iron-induced rat renal cell carcinoma

A growing number of studies indicate a link between oxidative stress and cancer. We previously developed a rat model of renal cell carcinoma (RCC) induced by ferric nitrilotriacetate (Fe-NTA). Here, we performed a genome-wide analysis to study characteristics of genomic alteration and identify putative genes involved in the development of Fe-NTA-induced RCCs. Array-based comparative genomic hybridization analyses revealed a chromosomal loss spanning chromosome 8 in most of the RCCs studied, with a common deletion at 8q31-32, which was confirmed by loss of heterozygosity (LOH) analysis. Studies of gene expression in RCCs or following Fe-NTA treatment revealed globally decreased transcription levels of 34 genes derived from chromosome 8 that are expressed in the kidney. Among them, the aminoacylase 1 (Acy1) gene, which maps to 8q32 and is highly expressed in the kidney, displayed a significantly decreased level of expression in RCCs. Significant amounts of the Acy1 protein were detected in the cytoplasm as well as in the nuclei of renal proximal tubular cells of untreated rats. Transfection of Acy1 into RCC cell lines inhibited proliferation and colony formation on soft agar. An increased number of apoptotic cells were observed following Acy1 transfection. The rat 8q31-32 chromosomal region corresponds to human 3p21.31-24.1, a hot spot where LOH is frequently found in various human cancers. Thus, Fe-NTA-induced renal tumor model is ideal for studying the link between deletions within this region and tumor formation. Our data demonstrate that Acy1 functions as a tumor suppressor in this rat RCC model.

Regulation of androgen receptor transactivity and mTOR-S6 kinase pathway by Rheb in prostate cancer cell proliferation.

Ras homolog‐enriched in brain (Rheb), a small GTP‐binding protein, is associated with prostate carcinogenesis through activating mammalian target of rapamycin (mTOR) signaling pathway. This study aimed to elucidate whether Rheb promotes proliferation of prostate cancer cells and can act as a potent therapeutic target in prostate cancer.

Primary carcinoid tumor arising in a retroperitoneal mature teratoma in an adult.

Abstract  An extremely rare case of a primary carcinoid tumor arising in a mature retroperitoneal teratoma is reported. A 53‐year‐old woman was admitted for further examination of an incidental retroperitoneal mass with calcification. Computed tomography scans demonstrated a tumor with fat, soft tissue and bone densities on the left renal hilum. Surgical excision of the tumor was performed with a preoperative diagnosis of retroperitoneal teratoma. The pathological diagnosis was mature teratoma, including all three germ layers. A carcinoid tumor was evident among teratoid tissues and it was thought to be a teratoma with malignant transformation. The patient did not have a carcinoid syndrome and had an uneventful recovery. She has been followed for 31 months with no recurrence. Carcinoid tumors rarely occur in teratomas of the ovary and the testis and, to our knowledge, this is the first case of carcinoid arising in a retroperitoneal mature teratoma.

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)

Exploring the optimal sequence of abiraterone and enzalutamide in patients with chemotherapy-naïve castration-resistant prostate cancer: The Kyoto-Baltimore collaboration

To evaluate and compare the efficacy of sequential treatment with abiraterone followed by enzalutamide or vice versa for castration‐resistant prostate cancer.

Decreased expression of lysophosphatidylcholine (16:0/OH) in high resolution imaging mass spectrometry independently predicts biochemical recurrence after surgical treatment for prostate cancer.

Human prostate cancers are highly heterogeneous, indicating a need for various novel biomarkers to predict their prognosis. Lipid metabolism affects numerous cellular processes, including cell growth, proliferation, differentiation, and motility. Direct profiling of lipids in tissue using high‐resolution matrix‐assisted laser desorption/ionization imaging mass spectrometry (HR‐MALDI‐IMS) may provide molecular details that supplement tissue morphology.