Tomomi Kamba

Identification of EP4 as a Potential Target for the Treatment of Castration-Resistant Prostate Cancer Using a Novel Xenograft Model

More effective therapeutic approaches for castration-resistant prostate cancer (CRPC) are urgently needed, thus reinforcing the need to understand how prostate tumors progress to castration resistance. We have established a novel mouse xenograft model of prostate cancer, KUCaP-2, which expresses the wild-type androgen receptor (AR) and which produces the prostate-specific antigen (PSA). In this model, tumors regress soon after castration, but then reproducibly restore their ability to proliferate after 1 to 2 months without AR mutation, mimicking the clinical behavior of CRPC. In the present study, we used this model to identify novel therapeutic targets for CRPC. Evaluating tumor tissues at various stages by gene expression profiling, we discovered that the prostaglandin E receptor EP4 subtype (EP4) was significantly upregulated during progression to castration resistance. Immunohistochemical results of human prostate cancer tissues confirmed that EP4 expression was higher in CRPC compared with hormone-naïve prostate cancer. Ectopic overexpression of EP4 in LNCaP cells (LNCaP-EP4 cells) drove proliferation and PSA production in the absence of androgen supplementation in vitro and in vivo. Androgen-independent proliferation of LNCaP-EP4 cells was suppressed when AR expression was attenuated by RNA interference. Treatment of LNCaP-EP4 cells with a specific EP4 antagonist, ONO-AE3-208, decreased intracellular cyclic AMP levels, suppressed PSA production in vitro, and inhibited castration-resistant growth of LNCaP-EP4 or KUCaP-2 tumors in vivo. Our findings reveal that EP4 overexpression, via AR activation, supports an important mechanism for castration-resistant progression of prostate cancer. Furthermore, they prompt further evaluation of EP4 antagonists as a novel therapeutic modality to treat CRPC.

ABCG2 421C>A polymorphism and high exposure of sunitinib in a patient with renal cell carcinoma

Sunitinib is an oral multitargeted tyrosine kinase inhibitor (TKI) for renal cell carcinoma (RCC) [1]. Due to severe adverse effects, patients are forced to reduce or discontinue sunitinib, and discontinuation often leads to tumor regrowth. Breast cancer resistance protein (ABCG2) expressed in the intestine functions as an efflux pump of drugs. Among single-nucleotide polymorphisms (SNPs) in the ABCG2 gene, ABCG2 421C>A is the most common mutant allele in Asians ( 30%) [2]. This SNP was reported to be associated with an increase in oral bioavailability of several drugs including TKIs [2]. In the clinical setting, we experienced one RCC patient with severe adverse events early after oral administration. We then carried out pharmacokinetic and pharmacogenetic analyses of sunitinib, focusing on ABCG2. Five RCC patients, three men and two women [mean age: 55.6 (34–67)], were analyzed. All five patients started 50 mg of sunitinib once a day. Adverse events were graded by National Cancer Institute—Common Toxicity Criteria for Adverse Effects v3.0. This study was approved by Kyoto University Graduate School and Faculty of Medicine Ethics Committee. Blood samples were collected on day 8, and plasma concentrations of sunitinib and its major metabolite SU012662 were determined by high-performance liquid chromatography (HPLC). HPLC conditions were as follows: column, GEMINI-NX 3 lm C18 (4.6 · 150 mm); mobile phase, acetate buffer in methanol at 33 : 67 (sunitinib) and 40 : 60 (SU012662) and wavelength, 423 nm. The SNP was determined by direct sequencing. Cell culture, transfection and transport experiments were carried out as described previously with a slight modification [3, 4]. One patient (patient 1) showed severe adverse events such as hypertension (grade 3), facial acne (grade 3) and elevation of amylase (grade 3). The maximum concentration and area under the concentration–time curve of sunitinib in patient 1 were 2.5-fold higher than in the other four patients (Figure 1A). Plasma concentration profiles of SU012662, a major metabolite of sunitinib, were comparable among patients (data not

Analysis of regulatory polymorphisms in organic ion transporter genes (SLC22A) in the kidney.

AbstractOrganic cation transporters (OCTs) and organic anion transporters (OATs) (SLC22A family) play crucial roles in the renal secretion of various drugs. Messengar ribonucleic acid (mRNA) expression of transporters can be a key factor regulating interindividual differences in drug pharmacokinetics. However, the source of variations in mRNA levels of transporters is unclear. In this study, we focused on single nucleotide polymorphisms (SNP) in the promoter region [regulatory SNPs (rSNPs)] as candidates for the factor regulating mRNA levels of SLC22A. We sequenced the promoter regions of OCT2 and OAT1–4 in 63 patients and investigated the effects of the identified rSNPs on transcriptional activities and mRNA expression. In the OCT2 promoter region, one deletion polymorphism (−578_−576delAAG) was identified; −578_−576delAAG significantly reduced OCT2 promoter activity (p < 0.05), and carriers of −578_−576delAAG tend to have lower OCT2 mRNA levels, but the difference is not significant. There was no rSNP in the OAT1 and OAT2 genes. The five rSNPs of OAT3 and one rSNP of OAT4 were unlikely to influence mRNA expression and promoter activity. This is the first study to investigate the influences of rSNPs on mRNA expression of SLC22A in the kidney and to identify a regulatory polymorphism affecting OCT2 promoter activity.

Outcome of different post-orchiectomy management for stage I seminoma: Japanese multi-institutional study including 425 patients

Objectives:  To clarify the contemporary clinical outcome of stage I seminoma and to provide information on treatment options to patients.

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.

Antiandrogen withdrawal syndrome and alternative antiandrogen therapy associated with the W741C mutant androgen receptor in a novel prostate cancer xenograft

The mechanisms underlying antiandrogen withdrawal syndrome (AWS) and alternative antiandrogen therapy (AAT) effectiveness were assumed to be mutations in the androgen receptor (AR), which resulted in an altered response to antiandrogens. The aim of the present study was to test this assumption using the novel prostate cancer xenograft model KUCaP‐1 harboring the W741C mutant AR (Yoshida et al., Cancer Res 2005; 65(21): 9611–9616).

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.

Prevalence of Skeletal Complications and Their Impact on Survival of Hormone Refractory Prostate Cancer Patients in Japan

OBJECTIVES Skeletal complications are a crucial factor in the quality of life and the prognosis of patients with hormone-refractory prostate cancer (HRPC); however, their true prevalence and impact on the prognosis remain largely unknown. In this study, we retrospectively investigated the prevalence of skeletal complications and survival of HRPC patients in a single Japanese institute. METHODS From 1990 to 2004, 151 patients with prostate cancer were estimated to have HRPC after androgen ablation therapy in a single institute. The medical records of the patients were reviewed for documentation of skeletal complications, and overall survival was calculated from the diagnosis of HRPC. RESULTS At the diagnosis of HRPC, bone metastases and bone pain were observed in 83.8% and 44.7% of patients, respectively. During follow-up, 80.0% and 14.2% of patients experienced bone pain and bone fracture, respectively. Moreover, 74.2% and 43.9% of the patients received nonsteroidal anti-inflammatory drugs and opioids for bone pain, respectively, and 50.9% of the patients underwent radiotherapy for palliative pain control. A high score on the extent of disease observed on the bone scan and the presence of bone pain at the diagnosis of HRPC were independent prognostic factors on multivariate analysis (hazard ratio = 1.753 and 2.118, P = .033 and .007, respectively). CONCLUSIONS Despite a small number of subjects, our retrospective survey demonstrated the high prevalence and mortality rate of skeletal complications in Japanese HRPC patients. Patients with bone pain at the diagnosis of HRPC had significantly poorer prognosis than those without.

Precise comparison of protein localization among OCT, OAT, and MATE in human kidney

Organic anion transporters (OATs) and organic cation transporters (OCT) play pivotal roles in the uptake of drugs into epithelial cells at the basolateral membranes, and multidrug and toxin extrusion (MATE) mediates drug secretion into urine at the brush-border membranes. In this study, the expression and distribution of apical MATE1 and MATE2-K, and basolateral OAT1, OAT3, and OCT2 were compared using serial sections of human kidney cortex. First, mRNA expression in the proximal tubules was evaluated using laser microdissection. Levels of OAT, OCT2, and MATE mRNA in the proximal tubules were greatly higher compared with glomerulus. The results quantitatively indicated that these transporters were localized to proximal tubules in the renal cortex. Second, MATE1 and MATE2-K protein were detected in proximal epithelial cells in which OCT2 protein was expressed at the basolateral membranes. In addition, MATE1 was expressed at the brush-border membranes of tubular epithelial cells in which OAT1 and OAT3 were expressed. The results confirmed that OAT1, OAT3, OCT2, MATE1, and MATE2-K were coexpressed in tubular epithelial cells. The cooperation among OAT, OCT, and MATE in renal drug secretion was consistent with their distribution.