CCL2 as a potential therapeutic target for clear cell renal cell carcinoma

We previously reported that the pVHL-atypical PKC-JunB pathway contributed to promotion of cell invasiveness and angiogenesis in clear cell renal cell carcinoma (ccRCC), and we detected chemokine (C-C motif) ligand-2 (CCL2) as one of downstream effectors of JunB. CCL2 plays a critical role in tumorigenesis in other types of cancer, but its role in ccRCC remains unclear. In this study, we investigated the roles and therapeutic potential of CCL2 in ccRCC. Immunohistochemical analysis of CCL2 expression for ccRCC specimens showed that upregulation of CCL2 expression correlated with clinical stage, overall survival, and macrophage infiltration. For functional analysis of CCL2 in ccRCC cells, we generated subclones of WT8 cells that overexpressed CCL2 and subclones 786-O cells in which CCL2 expression was knocked down. Although CCL2 expression did not affect cell proliferation in vitro, CCL2 overexpression enhanced and CCL2 knockdown suppressed tumor growth, angiogenesis, and macrophage infiltration in vivo. We then depleted macrophages from tumor xenografts by administration of clodronate liposomes to confirm the role of macrophages in ccRCC. Depletion of macrophages suppressed tumor growth and angiogenesis. To examine the effect of inhibiting CCL2 activity in ccRCC, we administered CCL2 neutralizing antibody to primary RCC xenografts established from patient surgical specimens. Inhibition of CCL2 activity resulted in significant suppression of tumor growth, angiogenesis, and macrophage infiltration. These results suggest that CCL2 is involved in angiogenesis and macrophage infiltration in ccRCC, and that CCL2 could be a potential therapeutic target for ccRCC

Functional and genomic characterization of patient‐derived xenograft model to study the adaptation to mTORC1 inhibitor in clear cell renal cell carcinoma

Resistance to the mechanistic target of rapamycin (mTOR) inhibitors, which are a standard treatment for advanced clear cell renal cell carcinoma (ccRCC), eventually develops in most cases. In this study, we established a patient-derived xenograft (PDX) model which acquired resistance to the mTOR inhibitor temsirolimus, and explored the underlying mechanisms of resistance acquisition. Temsirolimus was administered to PDX model mice, and one cohort of PDX models acquired resistance after repeated passages. PDX tumors were genetically analyzed by whole-exome sequencing and detected several genetic alterations specific to resistant tumors. Among them, mutations in ANKRD12 and DNMT1 were already identified in the early passage of a resistant PDX model, and we focused on a DNMT1 mutation as a potential candidate for developing the resistant phenotype. While DNMT1 expression in temsirolimus-resistant tumors was comparable with the control tumors, DNMT enzyme activity was decreased in resistant tumors compared with controls. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9-mediated heterozygous knockdown of DNMT1 in the temsirolimus-sensitive ccRCC (786-O) cell line was shown to result in a temsirolimus-resistant phenotype in vitro and in vivo. Integrated gene profiles using methylation and microarray analyses of PDX tumors suggested a global shift for the hypomethylation status including promotor regions, and showed the upregulation of several molecules that regulate the mTOR pathway in temsirolimus-resistant tumors. Present study showed the feasibility of PDX model to explore the mechanisms of mTOR resistance acquisition and suggested that genetic alterations, including that of DNMT1, which alter the methylation status in cancer cells, are one of the potential mechanisms of developing resistance to temsirolimus

CCL2は淡明型腎細胞癌に対する治療ターゲットとなりうる

京都大学0048新制・課程博士博士(医学)甲第20265号医博第4224号新制||医||1021(附属図書館)京都大学大学院医学研究科医学専攻(主査)教授 柳田 素子, 教授 武田 俊一, 教授 野田 亮学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDFA

間欠的内分泌療法は高分化型前立腺癌が内分泌不応性となるまでの期間を延長するかもしれない

われわれは過去にPSAが0.3ng/ml以下になれば休薬し2.0ng/ml以上になれば再開する方法で行った前立腺癌に対する間欠的内分泌療法の成績を発表した.今回は間欠療法と通常療法で内分泌不応性となるまでの期間を比較した.1995～2003年の間に限局性, 転移性前立腺癌と診断された患者および根治的前立腺全摘出後に生化学的再発をきたした46人の患者を間欠的内分泌療法で治療した.この中で間欠的内分泌療法の第2サイクル以降に入った患者(30人)を研究対象にし, 通常療法を受けPSAの底値が0.3ng/ml以下になった患者33人をコントロールとした.間欠療法, 通常療法の5年間生化学的非再発率はそれぞれ59%および89%で有意差はなかった(p=0.5).高分化型前立腺癌に対しては間欠療法が, 中低分化型に対しては通常療法が有意に5年間生化学的非再発率が高い結果となった.これらの結果より高分化型前立腺癌に対して間欠的内分泌療法は通常の方法よりも有効な治療法であることが示唆された(著者抄録)We previously reported the results of a pilot study of intermittent androgen deprivation (IAD) therapy in which surveillance was performed when PSA level fell below 0.3 ng/ml and androgen deprivation was resumed when PSA level exceeded 2.0 ng/ml. In the present study, we compared the duration of androgen dependence in patients treated with IAD with that in patients with continuous androgen deprivation (CAD) therapy. Forty-six patients with clinically localized or metastatic prostate cancer, or biochemical recurrence after radical prostatectomy were treated with IAD from 1995 to 2003. Patients in or after the second cycle of IAD (30 patients) were evaluated for duration of androgen dependence. Patients whose serum PSA nadir became <0.3 ng/ml (33 patients) represented a control group of CAD. The overall 5-year biochemical progression-free rate was 58% and 89% in the IAD and CAD groups, respectively; there was no significant difference between the two groups (p=0.5). Subgroup analysis showed that, irrespective of metastasis, the 5-year biochemical progression-free survival rate in the IAD group was not significantly different from that in the CAD group. However, IAD offered significantly better results for well-differentiated prostate cancer, whereas CAD offered significantly better results for moderately or poorly differentiated prostate cancer. The results obtained from this retrospective and nonrandomized study suggested that IAD may be a feasible treatment for well-differentiated prostate cancer

CCL

We previously reported that the pVHL-atypical PKC-JunB pathway contributed to promotion of cell invasiveness and angiogenesis in clear cell renal cell carcinoma (ccRCC), and we detected chemokine (C-C motif) ligand-2 (CCL2) as one of downstream effectors of JunB. CCL2 plays a critical role in tumorigenesis in other types of cancer, but its role in ccRCC remains unclear. In this study, we investigated the roles and therapeutic potential of CCL2 in ccRCC. Immunohistochemical analysis of CCL2 expression for ccRCC specimens showed that upregulation of CCL2 expression correlated with clinical stage, overall survival, and macrophage infiltration. For functional analysis of CCL2 in ccRCC cells, we generated subclones of WT8 cells that overexpressed CCL2 and subclones 786-O cells in which CCL2 expression was knocked down. Although CCL2 expression did not affect cell proliferation in vitro, CCL2 overexpression enhanced and CCL2 knockdown suppressed tumor growth, angiogenesis, and macrophage infiltration in vivo. We then depleted macrophages from tumor xenografts by administration of clodronate liposomes to confirm the role of macrophages in ccRCC. Depletion of macrophages suppressed tumor growth and angiogenesis. To examine the effect of inhibiting CCL2 activity in ccRCC, we administered CCL2 neutralizing antibody to primary RCC xenografts established from patient surgical specimens. Inhibition of CCL2 activity resulted in significant suppression of tumor growth, angiogenesis, and macrophage infiltration. These results suggest that CCL2 is involved in angiogenesis and macrophage infiltration in ccRCC, and that CCL2 could be a potential therapeutic target for ccRCC

Overexpression of IL13RA2 leads to acquired resistance to sunitinib and shRNA-mediated IL13RA2 knockdown induces sensitivity to sunitinib.

<p>(A) Immunoblot analysis of 786-O subclones infected with retrovirus encoding mock or WT IL13RA2. Whole cell extracts were immunoblotted using the indicated antibodies. Sequential changes in subcutaneous xenograft tumors from 786-O subclones infected with (B) mock or (C) WT IL13RA2 treated with sunitinib and vehicle (control). Each time point represents the mean ± SE of tumor volume in each group. The difference in tumor size between the treatment group and control was statistically significant in 786-O-mock cells but not statistically significant in 786-O-IL13RA2 cells (*<i>P</i> < 0.05, n.s.: not significant; two-way repeated ANOVA). The horizontal arrow bars indicate the periods of sunitinib administration. (D) Immunoblot analysis of Caki-1 subclones infected with lentivirus encoding scrambled or IL13RA2 shRNA. Whole cell extracts were immunoblotted using the indicated antibodies. Sequential changes of subcutaneous xenograft tumors from a Caki-1 subclone infected with (E) scrambled or (F) IL13RA2 shRNA treated with sunitinib and vehicle (control). Each time point represents the mean ± SE of tumor volume in each group. Day 0 is the first day of sunitinib administration 4 weeks after transplantation. The difference in tumor size between the treatment group and control was not significant in Caki-1-sh-scrambled cells but statistically significant in Caki-1-sh-IL13RA2 cells (n.s.: not significant, *<i>P</i> < 0.05; two-way repeated ANOVA). The arrow bars indicate the period of sunitinib administration.</p

Evaluation of IL13RA2 mRNA and protein expression.

<p>(A) Evaluation of IL13RA2 mRNA expression in KURC1 and KURC2 xenograft tumors treated with sunitinib or vehicle by qPCR. All samples were prepared in triplicate and data are presented as the mean ± SE from indicated number of samples. Columns, mean; bar, SE. The difference in the mRNA expression levels between the sunitinib-treated group and control or sensitive group in KURC1 was statistically significant (*<i>P</i> < 0.01; Students’ <i>t</i>-test). There was no significant difference in KURC2 groups. (B) Immunohistochemical staining of IL13RA2 in KURC1 xenograft tumors. Scale bar, 50 μm. (C) IL13RA2 expression in human ccRCC tumors with the response to sunitinib treatment evaluated by immunohistochemistry. ccRCC tumor samples were collected from patients prior to sunitinib treatment. Left: representative pictures of immunohistochemistry sections of tumors showing none, weak, or strong staining for IL13RA2. Right: ratio of IL13RA2 expression pattern and correlation of the response to sunitinib treatment. Scale bar, 100 μm.</p

Evaluation of apoptosis and microvessel density after sunitinib treatment in our primary xenograft models.

<p>(A) ssDNA staining and (B) CD31 staining of KURC1 treated with sunitinib or vehicle with different sensitivity status. Scale bar, 50 μm. Apoptosis was assessed by calculating the ssDNA positivity rate, and MVD was determined from CD31 staining using Image J software. Statistical analysis was performed using the Students’ <i>t</i>-test (*<i>P</i> < 0.01).</p

KURC1 tumors develop resistance to sunitinib but KURC2 remained sensitive.

<p>(A) Hematoxylin and eosin (H&E) staining of an original RCC surgical specimen and xenograft KURC1 and KURC2 tumor tissue. Scale bar, 50 μm. (B) The sequential changes of KURC1 and KURC2 xenograft tumors treated with sunitinib or vehicle only and (C) Left: KURC1 tumor volume at 4 weeks later from passage (sunitinib treatment day 0). Right: The sequential changes of KURC1 sunitinib 5^th and vehicle 5^th. KURC1 repeatedly treated with sunitinib or vehicle 5^th. Each time point represents the mean ± SE of tumor volume in each group. Day 0 is the first day of sunitinib administration at 4 weeks after transplantation. The difference in tumor size between sunitinib 5^th group and vehicle 5^th group in KURC1 was not statistically significant using two-way repeated ANOVA. Arrowed bars indicate the periods of sunitinib administration. ▼ indicates the time point when tumors were resected.</p