CCAR1 promotes chromatin loading of androgen receptor (AR) transcription complex by stabilizing the association between AR and GATA2.

Androgen receptor (AR), a ligand-dependent transcription factor, plays a critical role in prostate cancer onset and progression, and its transcriptional function is mediated largely by distinct nuclear receptor co-regulators. Here, we show that cell cycle and apoptosis regulator 1 (CCAR1) functions as an AR co-activator. CCAR1 interacted with and enhanced the transcriptional activity of AR. Depletion of CCAR1 caused reduction in androgen-dependent expression of a subset of AR target genes. We further showed that CCAR1 is required for recruitment of AR, MED1 and RNA polymerase II to the enhancers of AR target genes and for androgen-induced long-range prostate specific antigen enhancer-promoter interaction. The molecular mechanism underlying CCAR1 function in AR-mediated transcription involves CCAR1-mediated enhanced recruitment of GATA2, a pioneer factor for AR, to AR-binding sites. CCAR1 stabilized the interaction between AR and GATA2 by interacting directly with both proteins, thereby facilitating AR and GATA2 occupancy on the enhancers. Furthermore, CCAR1 depletion inhibited the growth, migration, invasion of prostate cancer cells and reduced the tumorigenicity of prostate cancer cells in vivo. Our results firmly established CCAR1 as an AR co-activator that plays a key role in AR transcription complex assembly and has an important physiological role in androgen signaling and prostate tumorigenesis.

Therapeutic Potential of Bipolar Androgen Therapy for Castration-Resistant Prostate Cancer: In Vitro and In Vivo Studies.

Androgen deprivation therapy (ADT) is a primary treatment for advanced prostate cancer (PCa), but resistance often leads to castration-resistant PCa (CRPC). CRPC remains androgen receptor (AR)-dependent, and AR overexpression causes vulnerability to high doses of androgen in CRPC. Bipolar androgen therapy (BAT) refers to the periodic administration of testosterone, resulting in oscillation between supraphysiologic and near-castrate serum testosterone levels. In this study, we evaluated the efficacy of BAT against CRPC in a preclinical setting. To emulate CRPC characteristics, PCa cell lines (LNCaP, VCaP, and 22Rv1) were cultured in phenol red-free RPMI-1640 medium supplemented with 10% dextran-coated charcoal treated FBS (A- cell line). Cell viability, AR, and AR-V7 expression were evaluated using the Cell Counting Kit-8 and Western blotting. In vivo studies involved 12 castrated NOG mice injected with LNCaP/A- cells, treated with testosterone pellets or controls in 2-week cycles. Tumor sizes were measured post a 6-week treatment cycle. Bicalutamide inhibited PCa cell viability but not in the adapted cell lines. Supraphysiologic androgen levels suppressed AR-expressing PCa cell growth in vitro. In vivo, high AR-expressing LNCaP cells proliferated under castrate conditions, while BAT-treated xenografts exhibited significant growth inhibition with low Ki-67 and mitotic indexes and a high cell death index. This study provides preliminary evidence that BAT is effective for the treatment of CRPC through rapid cycling between supraphysiologic and near-castrate serum testosterone levels, inducing an anti-tumor effect.

Evaluating a combination treatment of NK cells and reovirus against bladder cancer cells using an in vitro assay to simulate intravesical therapy.

Intravesical treatment using either reovirus or natural killer (NK) cells serves as an efficient strategy for the treatment of bladder cancer cells (BCCs); however, corresponding monotherapies have often shown modest cytotoxicity. The potential of a locoregional combination using high-dose reovirus and NK cell therapy in an intravesical approach has not yet been studied. In this study, we evaluated the effectiveness of reoviruses and expanded NK cells (eNK) as potential strategies for the treatment of bladder cancer. The anti-tumor effects of mono-treatment with reovirus type 3 Dearing strain (RC402 and RP116) and in combination with interleukin (IL)-18/-21-pretreated eNK cells were investigated on BCC lines (5637, HT-1376, and 253J-BV) using intravesical therapy to simulate in vitro model. RP116 and IL-18/-21-pretreated eNK cells exhibited effective cytotoxicity against grade 1 carcinoma (5637 cells) when used alone, but not against HT-1376 (grade 2 carcinoma) and 253J-BV cells (derived from a metastatic site). Notably, combining RP116 with IL-18/-21-pretreated eNK cells displayed effective cytotoxicity against both HT-1376 and 253J-BV cells. Our findings underscore the potential of a combination therapy using reoviruses and NK cells as a promising strategy for treating bladder cancer.

Development and characterization of a bladder cancer xenograft model using patient-derived tumor tissue.

Most of the cancer xenograft models are derived from tumor cell lines, but they do not sufficiently represent clinical cancer characteristics. Our objective was to develop xenograft models of bladder cancer derived from human tumor tissue and characterize them molecularly as well as histologically. A total of 65 bladder cancer tissues were transplanted to immunodeficient mice. Passagable six cases with clinico-pathologically heterogeneous bladder cancer were selected and their tumor tissues were collected (012T, 025T, 033T, 043T, 048T, and 052T). Xenografts were removed and processed for the following analyses: (i) histologic examination, (ii) short tandem repeat (STR) genotyping, (iii) mutational analysis, and (iv) array-based comparative genomic hybridization (array-CGH). The original tumor tissues (P 0) and xenografts of passage 2 or higher (≥P2) were analyzed and compared. As a result, hematoxylin and eosin staining revealed the same histologic architecture and degree of differentiation in the primary and xenograft tumors in all six cases. Xenograft models 043T_P2 and 048T_P2 had completely identical STR profiles to the original samples for all STR loci. The other models had nearly identical STR profiles. On mutational analysis, four out of six xenografts had mutations identical to the original samples for TP53, HRAS, BRAF, and CTNNB1. Array-CGH analysis revealed that all six xenograft models had genomic alterations similar to the original tumor samples. In conclusion, our xenograft bladder cancer model derived from patient tumor tissue is expected to be useful for studying the heterogeneity of the tumor populations in bladder cancer and for evaluating new treatments.

Chromosomal Instability in Cell-free DNA as a Prognostic Biomarker of Metastatic Hormone-sensitive Prostate Cancer Treated with Androgen Deprivation Therapy.

BACKGROUND: Although patients with metastatic hormone-sensitive prostate cancer (mHSPC) undergo androgen deprivation therapy (ADT), the disease can progress to metastatic castration-resistant prostate cancer (mCRPC). There are no reliable biomarkers for predicting this progression. Chromosomal instability resulting in copy number alterations (CNAs) is characteristically observed in patients with various cancers. OBJECTIVE: To investigate the role of chromosomal instability in patients with mHSPC. DESIGN, SETTING, AND PARTICIPANTS: This prospective study analyzed cell-free DNA (cfDNA) in pretreatment plasma samples from 75 patients with elevated prostate-specific antigen. Low-depth whole-genome sequencing of cfDNA was performed to identify CNAs. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The I score (sum of the product of the absolute Z score and the corresponding chromosome length) was used as a measure of chromosomal instability. Kaplan-Meier and Cox proportional-hazard regression analyses were performed to evaluate the association between the I score and time to progression (TTP) and the prognostic value of chromosomal instability in predicting castration resistance, respectively. RESULTS AND LIMITATIONS: Of 22 patients with a positive I score, 86.4% (19/22) had metastatic prostate cancer. Of these 19 cases, 94.7% (18/19) were mHSPC, which was high-volume mHSPC in 83.3% (15/18). None of the patients with localized prostate cancer had a positive I score. TTP in patients with mHSPC was significantly shorter in the positive than in the negative I-score group (16.4 vs 36.9 mo; p = 0.001). Only the I score could independently predict mCRPC development (hazard ratio 10.315, 95% confidence interval 1.141-93.208; p = 0.038). CONCLUSIONS: The I score could be a biomarker for ADT response and progression to mCRPC in patients with mHSPC. PATIENT SUMMARY: We investigated whether genetic changes in cell-free DNA can predict outcomes for patients with metastatic prostate cancer that still responds to hormone therapy. We found that chromosomal instability could be a potential predictor of the development of metastatic castration-resistant prostate cancer.

Genomic mutation profiling using liquid biopsy in Korean patients with prostate cancer: Circulating tumor DNA mutation predicts the development of castration resistance.

PURPOSE: To investigate germline and somatic mutation profiles in Korean patients with prostate cancer using liquid biopsy and solid tissue testing and to evaluate the prognostic value of circulating tumor DNA (ctDNA) in predicting castration resistance in patients with metastatic hormone-sensitive prostate cancer (mHSPC). MATERIALS AND METHODS: Plasma samples from 56 prostate cancer patients were subjected to next-generation sequencing (NGS) to identify germline mutations and ctDNA analysis using liquid biopsy to detect somatic mutations. Additionally, paired solid cancer tissues from 18 patients were subject to NGS to detect somatic mutations. The clinical parameters and ctDNA profiles of patients with mHSPC were analyzed to evaluate the prognostic value of ctDNA mutations with respect to predicting castration resistance using Cox proportional hazards regression analysis. RESULTS: Germline mutations occurred in 3.6% of the patients in this cohort, with mutations identified in RAD50 (1.8%) and BRCA1 (1.8%). Somatic mutations detected by liquid biopsy and solid tissue testing were common in TP53 (12.5%), PIK3CA (3.6%), and TMPRSS2-ERG (3.6%). Of the 18 patients with paired tissue testing, two patients had at least one identical somatic mutation in both the liquid biopsy and solid tissue testing. In patients with mHSPC, the presence of ctDNA mutations could independently predict the castration resistance development (hazard ratio, 13.048; 95% confidential interval, 1.109-153.505; p=0.041). CONCLUSIONS: Korean patients with prostate cancer showed a relatively low germline mutation rate compared to other ethnicities. The ctDNA mutations detected by liquid biopsy can predict the development of castration resistance in patients with mHSPC.

Bruceantin targets HSP90 to overcome resistance to hormone therapy in castration-resistant prostate cancer.

Rationale: Aberrant androgen receptor (AR) signaling via full-length AR (AR-FL) and constitutively active AR variant 7 (AR-V7) plays a key role in the development of castration-resistant prostate cancer (CRPC) and resistance to hormone therapies. Simultaneous targeting of AR-FL and AR-V7 may be a promising strategy to overcome resistance to hormone therapy. This study aimed to identify novel drug candidates co-targeting AR-FL and AR-V7 activities and elucidate their molecular mechanism of anti-CRPC activities. Methods: Using a CRPC cell-based reporter assay system, we screened a small library of antimalarial agents to explore the possibility of repositioning them for CRPC treatment and identified bruceantin (BCT) as a potent anti-CRPC drug candidate. A series of cell-based, molecular, biochemical, and in vivo approaches were performed to evaluate the therapeutic potential and molecular mechanism of BCT in CRPC. These approaches include reporter gene assays, cell proliferation, RNA-seq, qRT-PCR, mouse xenografts, co-immunoprecipitation, GST pull-down, immobilized BCT pull-down, molecular modeling, and bioinformatic analyses. Results: We identified BCT as a highly potent inhibitor co-targeting AR-FL and AR-V7 activity. BCT inhibits the transcriptional activity of AR-FL/AR-V7 and downregulates their target genes in CRPC cells. In addition, BCT efficiently suppresses tumor growth and metastasis of CRPC cells. Mechanistically, BCT disrupts the interaction of HSP90 with AR-FL/AR-V7 by directly binding to HSP90 and inhibits HSP90 chaperone function, leading to degradation of AR-FL/AR-V7 through the ubiquitin-proteasome system. Clinically, HSP90 expression is upregulated and correlated with AR/AR-V7 levels in CRPC. Conclusion: Our findings suggest that BCT could serve as a promising therapeutic candidate against CRPC and highlight the potential benefit of targeting AR-FL/AR-V7-HSP90 axis to overcome resistance caused by aberrant AR-FL/AR-V7 signaling.

Single-cell RNA sequencing reveals the tumor microenvironment and facilitates strategic choices to circumvent treatment failure in a chemorefractory bladder cancer patient.

BACKGROUND: Tumor cell-intrinsic mechanisms and complex interactions with the tumor microenvironment contribute to therapeutic failure via tumor evolution. It may be possible to overcome treatment resistance by developing a personalized approach against relapsing cancers based on a comprehensive analysis of cell type-specific transcriptomic changes over the clinical course of the disease using single-cell RNA sequencing (scRNA-seq). METHODS: Here, we used scRNA-seq to depict the tumor landscape of a single case of chemo-resistant metastatic, muscle-invasive urothelial bladder cancer (MIUBC) addicted to an activating Harvey rat sarcoma viral oncogene homolog (HRAS) mutation. In order to analyze tumor evolution and microenvironmental changes upon treatment, we also applied scRNA-seq to the corresponding patient-derived xenograft (PDX) before and after treatment with tipifarnib, a HRAS-targeting agent under clinical evaluation. RESULTS: In the parallel analysis of the human MIUBC and the PDX, diverse stromal and immune cell populations recapitulated the cellular composition in the human and mouse tumor microenvironment. Treatment with tipifarnib showed dramatic anticancer effects but was unable to achieve a complete response. Importantly, the comparative scRNA-seq analysis between pre- and post-tipifarnib-treated PDX revealed the nature of tipifarnib-refractory tumor cells and the tumor-supporting microenvironment. Based on the upregulation of programmed death-ligand 1 (PD-L1) in surviving tumor cells, and the accumulation of multiple immune-suppressive subsets from post-tipifarnib-treated PDX, a PD-L1 inhibitor, atezolizumab, was clinically applied; this resulted in a favorable response from the patient with acquired resistance to tipifarnib. CONCLUSION: We presented a single case report demonstrating the power of scRNA-seq for visualizing the tumor microenvironment and identifying molecular and cellular therapeutic targets in a treatment-refractory cancer patient.

DBC1 promotes castration-resistant prostate cancer by positively regulating DNA binding and stability of AR-V7.

Constitutively active AR-V7, one of the major androgen receptor (AR) splice variants lacking the ligand-binding domain, plays a key role in the development of castration-resistant prostate cancer (CRPC) and anti-androgen resistance. However, our understanding of the regulatory mechanisms of AR-V7-driven transcription is limited. Here we report DBC1 as a key regulator of AR-V7 transcriptional activity and stability in CRPC cells. DBC1 functions as a coactivator for AR-V7 and is required for the expression of AR-V7 target genes including CDH2, a mesenchymal marker linked to CRPC progression. DBC1 is required for recruitment of AR-V7 to its target enhancers and for long-range chromatin looping between the CDH2 enhancer and promoter. Mechanistically, DBC1 enhances DNA-binding activity of AR-V7 by direct interaction and inhibits CHIP E3 ligase-mediated ubiquitination and degradation of AR-V7 by competing with CHIP for AR-V7 binding, thereby stabilizing and activating AR-V7. Importantly, DBC1 depletion suppresses the tumorigenic and metastatic properties of CRPC cells. Our results firmly establish DBC1 as a critical AR-V7 coactivator that plays a key role in the regulation of DNA binding and stability of AR-V7 and has an important physiological role in CRPC progression.

Novel therapeutic roles of MC-4 in combination with everolimus against advanced renal cell carcinoma by dual targeting of Akt/pyruvate kinase muscle isozyme M2 and mechanistic target of rapamycin complex 1 pathways.

Current clinical trials of new anticancer therapies against metastatic renal cell carcinoma (RCC), including molecular-targeted therapies, have not shown promise. The purpose of this study was to preclinically assess the antitumor effects of MC-4, a partially purified material of Artemisia annua L., as a monotherapy or in combination with the known mechanistic target of rapamycin complex 1 (mTORC1) inhibitor, everolimus, against Caki-1 (Von Hippel-Lindau (VHL)+/+) and 786-O (VHL-/-) human RCC cells. MC-4 monotherapy significantly increased tumor growth inhibition and autophagic cell death in RCC cells in vitro and in vivo. Everolimus led to compensatory Akt activation by inhibiting only mTORC1 signaling pathway. In contrast to everolimus, MC-4 enhanced phosphatase and tensin homolog expression and reduced its downstream effector, Akt/pyruvate kinase muscle isozyme M2 (PKM2), leading to decreased expression of glucose transporter 1, which is associated with cancer cell metabolism. The synergistic antitumor and anti-metastatic effects induced by co-administration of MC-4 and everolimus involve cell growth inhibition and autophagic cell death via dual targeting of phosphatidylinositol 3-kinase (PI3K)/Akt/PKM2 and mTORC1. These findings suggest that MC-4 is a novel Akt/PKM2 inhibitor that can overcome the limitation of existing mTOR inhibitors and can be considered a novel strategy to treat patients with rapidly progressing advanced RCC.

Enhancement of in vivo endothelialization of tissue-engineered vascular grafts by granulocyte colony-stimulating factor.

Successful reconstruction of large-diameter blood vessel in humans has been demonstrated using the tissue engineering technique, but improvement in patency of small-diameter bioartificial vascular graft remains a great challenge. This study reports that granulocyte colony-stimulating factor (G-CSF) can enhance in vivo endothelialization of tissue-engineered vascular grafts, which could be used to improve patency of small-diameter vascular graft. Vascular grafts were tissue engineered with decellularized canine abdominal aortas and canine autologous bone marrow-derived cells. Prior to cell seeding onto decellularized graft matrices, bone marrow-derived cells were induced to differentiate into endothelial cells and smooth muscle cells. The cell-seeded vascular grafts were implanted into the abdominal aortas of bone marrow donor dogs. Before and after graft implantation, G-CSF was administered subcutaneously to the dogs (n = 3). The grafts implanted into the dogs not receiving G-CSF were used as controls (n = 3). Eight weeks after implantation, grafts in both groups showed regeneration of vascular tissues including endothelium and smooth muscle. Importantly, endothelium formation was more extensive in the G-CSF-treated grafts than in the control grafts, as assessed with reverse transcription polymerase chain reaction, western blot, and immunohistochemistry. In addition, intimal hyperplasia was significantly reduced in the G-CSF-treated grafts compared to the control grafts. This study suggests that G-CSF administration could be applied to improve patency of small-diameter tissue-engineered vascular grafts.

Curcumin potentiates antitumor activity of cisplatin in bladder cancer cell lines via ROS-mediated activation of ERK1/2.

Resistance of bladder cancer to cisplatin is a major obstacle to successful treatment. In the current study, we investigated the apoptotic effects of curcumin and cisplatin co-treatment in 253J-Bv(p53 wild-type) and T24(p53 mutant) bladder cancer. We found that curcumin and cisplatin co-treatment primarily targets reactive oxygen species(ROS) and extracellular regulated kinase(ERK) signaling during the apoptosis induction in bladder cancer. The apoptosis rate in 253J-Bv and T24 cells co-treated with curcumin and cisplatin was increased compared to that in cells exposed to single-agent treatment conditions. Also, caspase-3 activation and ROS production were observed in both cells treated with curcumin and cisplatin, together with upregulation of p-MEK and p-ERK1/2 signaling. NAC(ROS scavenger) and U0126(ERK inhibitor) inhibited apoptosis induced by curcumin and cisplatin. In addition, when 253J-Bv cells were co-treated with curcumin and cisplatin, p53 and p21 expression levels were markedly increased when compared to controls. Unlike 253J-Bv cells, T24 cells were co-treated with curcumin and cisplatin revealed an induction of apoptosis through decreased p-signal transducer and activator of transcription 3(STAT3) expression. Moreover, pretreatment with U0126 suppressed curcumin and cisplatin-induced upregulation of p53, p21, and p-STAT3 and downregulation of survival proteins in both cells. In conclusion, co-treatment with curcumin and cisplatin synergistically induced apoptosis through ROS-mediated activation of ERK1/2 in bladder cancer.

Immunohistochemical staining of ERG and SOX9 as potential biomarkers of docetaxel response in patients with metastatic castration-resistant prostate cancer.

We aimed to evaluate ERG and SOX9 as potential biomarkers of docetaxel response in metastatic castration-resistant prostate cancer (mCRPC) patients. Seventy-one mCRPC patients were evaluated. Tissue microarrays were constructed and immunohistochemistry was performed. Treatment response was assessed by prostate specific antigen (PSA) response rate, PSA progression-free survival (PSA-PFS), clinical/radiologic PFS (C/R-PFS) and overall survival (OS). ERG and SOX9 were found in 13 (18.3%) and 62 (87.3%) patients, respectively. ERG-positive had lower PSA response rates than negative (15.4% vs 62.1%, p = 0.004), and SOX9 showed a same trend (46.8% vs 100.0%, p = 0.003). ERG positivity correlated with a lower PSA-PFS (3.2 mos vs 7.4 mos, p < 0.001), C/R-PFS (3.8 mos vs 9.0 mos, p < 0.001) and OS (10.8 mos vs 21.4 mos, p < 0.001). SOX9 positivity also showed a lower PSA-PFS, C/R-PFS and OS (p =0.006, p =0.012 and p =0.023, respectively). On multivariate analysis, ERG positivity was a significant risk factor for a lower PSA-PFS, C/R-PFS and OS (p < 0.001, p < 0.001 and p =0.001, respectively). SOX9 expression was also a risk factor for a lower PSA-PFS, C/R-PFS and OS (p = 0.018, p = 0.025 and p =0.047, respectively). These findings indicate that ERG and SOX9 is potential biomarkers for prediction to docetaxel treatment in mCRPC patients.

Establishment and antitumor effects of dasatinib and PKI-587 in BD-138T, a patient-derived muscle invasive bladder cancer preclinical platform with concomitant EGFR amplification and PTEN deletion.

Muscle-invasive bladder cancer (MIBC) consists of a heterogeneous group of tumors with a high rate of metastasis and mortality. To facilitate the in-depth investigation and validation of tailored strategies for MIBC treatment, we have developed an integrated approach using advanced high-throughput drug screening and a clinically relevant patient-derived preclinical platform. We isolated patient-derived tumor cells (PDCs) from a rare MIBC case (BD-138T) that harbors concomitant epidermal growth factor receptor (EGFR) amplification and phosphatase and tensin homolog (PTEN) deletion. High-throughput in vitro drug screening demonstrated that dasatinib, a SRC inhibitor, and PKI-587, a dual PI3K/mTOR inhibitor, exhibited targeted anti-proliferative and pro-apoptotic effects against BD-138T PDCs. Using established patient-derived xenograft models that successfully retain the genomic and molecular characteristics of the parental tumor, we confirmed that these anti-tumor responses occurred through the inhibition of SRC and PI3K/AKT/mTOR signaling pathways. Taken together, these experimental results demonstrate that dasatinib and PKI-587 might serve as promising anticancer drug candidates for treating MIBC with combined EGFR gene amplification and PTEN deletion.

Repurposing the anti-malarial drug artesunate as a novel therapeutic agent for metastatic renal cell carcinoma due to its attenuation of tumor growth, metastasis, and angiogenesis.

Despite advances in the development of molecularly targeted therapies, metastatic renal cell carcinoma (RCC) is still incurable. Artesunate (ART), a well-known anti-malarial drug with low toxicity, exhibits highly selective anti-tumor actions against various tumors through generation of cytotoxic carbon-centered free radical in the presence of free iron. However, the therapeutic efficacy of ART against metastatic RCC has not yet been fully elucidated. In the analysis on a dataset from The Cancer Genome Atlas (TCGA) (n = 469) and a tissue microarray set from Samsung Medical Center (n = 119) from a cohort of patients with clear cell RCC (ccRCC), up-regulation of transferrin receptor 1 (TfR1), which is a well-known predictive marker for ART, was correlated with the presence of distant metastasis and an unfavorable prognosis. Moreover, ART exerted potent selective cytotoxicity against human RCC cell lines (Caki-1, 786-O, and SN12C-GFP-SRLu2) and sensitized these cells to sorafenib in vitro, and the extent of ART cytotoxicity correlated with TfR1 expression. ART-mediated growth inhibition of human RCC cell lines was shown to result from the induction of cell cycle arrest at the G2/M phase and oncosis-like cell death. Furthermore, ART inhibited cell clonogenicity and invasion of human RCC cells and anti-angiogenic effects in vitro in a dose-dependent manner. Consistent with these in vitro data, anti-tumor, anti-metastatic and anti-angiogenic effects of ART were also validated in human 786-O xenografts. Taken together, ART is a promising novel candidate for treating human RCC, either alone or in combination with other therapies.

Tpl2 kinase impacts tumor growth and metastasis of clear cell renal cell carcinoma.

UNLABELLED: Due to the innate high metastatic ability of renal cell carcinoma (RCC), many patients with RCC experience local or systemic relapses after surgical resection. A deeper understanding of the molecular pathogenesis underlying advanced RCC is essential for novel innovative therapeutics. Tumor progression locus 2 (Tpl2), upregulated in various tumor types, has been reported to be associated with oncogenesis and metastatic progression via activation of the MAPK signaling pathway. Herein, the relevance of Tpl2 in tumor growth and metastasis of RCC is explored. Inspection of The Cancer Genome Atlas (TCGA) indicated that Tpl2 overexpression was significantly related to the presence of metastases and poor outcome in clear cell RCC (ccRCC), which is the most aggressive subtype of RCC. Moreover, expression of Tpl2 and CXCR4 showed a positive correlation in ccRCC patients. Depletion of Tpl2 by RNAi or activity by a Tpl2 kinase inhibitor in human ccRCC cells remarkably suppressed MAPK pathways and impaired in vitro cell proliferation, clonogenicity, anoikis resistance, migration, and invasion capabilities. Similarly, orthotopic xenograft growth and lung metastasis were significantly inhibited by Tpl2 silencing. Furthermore, Tpl2 knockdown reduced CXCL12-directed chemotaxis and chemoinvasion accompanied with impaired downstream signaling, indicating potential involvement of Tpl2 in CXCR4-mediated metastasis. Taken together, these data indicate that Tpl2 kinase is associated with and contributes to disease progression of ccRCC. IMPLICATIONS: Tpl2 kinase activity has prognostic and therapeutic targeting potential in aggressive clear cell renal cell carcinoma.

Preliminary experience with tissue engineering of a venous vascular patch by using bone marrow-derived cells and a hybrid biodegradable polymer scaffold.

OBJECTIVE: Currently available synthetic polymer vascular patches used in cardiovascular surgery have shown serious shortcomings, including thrombosis, calcification, infection, and lack of growth potential. These problems may be avoided by vascular patches tissue-engineered with autologous stem cells and biodegradable polymeric materials. The objective of this study was to develop a tissue-engineered vascular patch by using autologous bone marrow-derived cells (BMCs) and a hybrid biodegradable polymer scaffold. METHODS: Hybrid biodegradable polymer scaffolds were fabricated from poly(lactide-co-epsilon-caprolactone) (PLCL) copolymer reinforced with poly(glycolic acid) (PGA) fibers. Canine bone marrow mononuclear cells were induced in vitro to differentiate into vascular smooth muscle cells and endothelial cells. Tissue-engineered vascular patches (15 mm wide x 30 mm long) were fabricated by seeding vascular cells onto PGA/PLCL scaffolds and implanted into the inferior vena cava of bone marrow donor dogs. RESULTS: Compared with PLCL scaffolds, PGA/PLCL scaffolds exhibited tensile mechanical properties more similar to those of dog inferior vena cava. Eight weeks after implantation of vascular patches tissue-engineered with BMCs and PGA/PLCL scaffolds, the vascular patches remained patent with no sign of thrombosis, stenosis, or dilatation. Histological, immunohistochemical, and scanning electron microscopic analyses of the retrieved vascular patches revealed regeneration of endothelium and smooth muscle, as well as the presence of collagen. Calcium deposition on tissue-engineered vascular patches was not significantly different from that on native blood vessels. Immunofluorescent double staining confirmed that implanted BMCs survived after implantation and contributed to regeneration of endothelium and vascular smooth muscle in the implanted vascular patches. CONCLUSIONS: This study demonstrates that vascular patches can be tissue-engineered with autologous BMCs and hybrid biodegradable polymer scaffolds.

Angiogenesis facilitated by autologous whole bone marrow stem cell transplantation for Buerger's disease.

We hypothesized that angiogenesis can be triggered by autologous whole bone marrow stem cell transplantation. Twenty-seven patients (34 lower limbs) with Buerger's disease, who were not candidates for surgical revascularization or radiologic intervention, were enrolled in this study. Six sites of the tibia bone were fenestrated using a 2.5-mm-diameter screw under fluoroscopic guidance. Clinical status and outcome were determined using the "Recommended Standards for Reports." To mobilize endothelial progenitor cells (EPCs) from bone marrow, recombinant human granulocyte colony-stimulating factor (r-HuG-CSF) was injected subcutaneously as a dose of 75 microg, preoperatively. During the follow-up period (19.1 +/- 3.5 months), one limb showed a markedly improved outcome (+3), and 26 limbs showed a moderately improved outcome (+2). Thirteen limbs among 17 limbs with nonhealing ulcers healed. Postoperative angiograms were obtained for 22 limbs. Two limbs showed marked (+3), five limbs moderate (+2), and nine limbs slight (+1) collateral development. However, six limbs showed no collateral development (0). Peripheral blood and bone marrow samples were analyzed for CD34 and CD133 molecules to enumerate potential EPCs, and EPC numbers were found to be increased in peripheral blood and in bone marrow after r-HuG-CSF injection. In conclusion, the transplantation of autologous whole BMCs by fenestration of the tibia bone represents a simple, safe, and effective means of inducing therapeutic angiogenesis in patients with Buerger's disease.