Primary Renal Cell Carcinoma Tissues Research Articles

Epigenetic silencing of ZBTB28 promotes renal cell carcinogenesis.

Zinc finger and BTB domain-containing protein 28 (ZBTB28) is a potential tumor suppressor for some cancers. However, its epigenetic regulation and functions in renal cell carcinoma (RCC) remain to be elucidated. The expression of ZBTB28 mRNA was analyzed by semi-quantitative reverse transcription polymerase chain reaction (PCR) in nine RCC cell lines and normal kidney tissues. Methylation status of ZBTB28 promoter was assessed by methylation-specific PCR in RCC cell lines, primary RCC, tumors and adjacent tissues. The involvement of ZBTB28 in cell proliferation and migration was investigated. ZBTB28 promoter was hypermethylated in 88.9% (8/9) of RCC cell lines with reduced ZBTB28 mRNA expression, and could be reversed by DNA methyltransferase inhibitors. The methylation of ZBTB28 promoter was detected in 73.5% (36/49) of primary RCC tissues, compared with 7.1% (1/14) in normal tissues. Overexpression of ZBTB28 significantly inhibited RCC cell proliferation and migration, and induced apoptosis. Further analyses revealed that ZBTB28 upregulation could inhibit multiple oncogenic signaling transduction pathways. ZBTB28 is frequently silenced by promoter methylation in RCC pathogenesis and functions as a novel tumor suppressive gene. ZBTB28 may be a potential target for the development of RCC therapeutic strategies.

Tuftelin 1 (TUFT1) Promotes the Proliferation and Migration of Renal Cell Carcinoma via PI3K/AKT Signaling Pathway.

Tuftelin 1 (TUFT1), a protein functioning distinctively in different tissues, is reported to be elevated in several types of cancers and the elevation of TUFT1 is correlated with unfavorable clinicopathologic characteristics and poor survival. However, the involvement of TUFT1 in renal cell carcinoma (RCC) remains unknown. In the current study, we investigated the role of TUFT1 in RCC and potential underlying mechanisms. RT-PCR and Western blot analysis showed that both the mRNA and protein levels of TUFT1 were increased in primary RCC tissue and RCC cell lines. TUFT1 overexpression in RCC cells resulted in enhanced cell proliferation and migration while knockdown of TUFT1 by contrast decreased the growth and migration of the RCC cells, indicating TUFT1 expression is involved in RCC cell growth and migration. The involvement of TUFT1 in the epithelial-mesenchymal transition (EMT) of RCC cells was also determined by measuring the expression of EMT-related markers. Our data showed that TUFT1 overexpression promoted RCC cell EMT progression while knockdown of TUFT1 suppressed such process. Further signaling pathway inhibition assay revealed that TUFT1-induced RCC cell growth, migration and EMT was significantly suppressed by PI3K inhibitor, but not JNK or MEK inhibitors. In addition, TUFT1 overexpression enhanced the AKT phosphorylation, a key member of the PI3K signaling pathway, while PI3K inhibitor suppressed such process. Taken together, our study showed that TUFT1 expression was elevated in RCC and such elevation promoted the proliferation, migration and EMT of RCC cells in vitro, through PI3K/AKT signaling pathway. The findings of our current study imply that TUFT1 is involved in RCC tumorigenesis, and it may serve as a biomarker for RCC diagnosis and a potential target for RCC treatment.

Alteration of CYP4A11 expression in renal cell carcinoma: diagnostic and prognostic implications.

Background: Cytochrome P-450 4A11 (CYP4A11) and peroxisome proliferator-activated receptor-α (PPARα) are expressed at high levels in renal proximal tubules, and upregulation of CYP4A11 protein levels is known to be influenced by PPAR agonists. The goal of this study was to evaluate the clinicopathological role of CYP4A11 expression in renal cell carcinoma (RCC).Methods: We performed immunohistochemical analysis of CYP4A11, CYP4A22 and PPARα and correlated the results with the clinicopathological features of RCC (n=139). Reverse transcription digital droplet polymerase chain reaction (RT-ddPCR) against CYP4A11 and CYP4A22 was also performed.Results: CYP4A11 mRNA expression levels were higher in non-neoplastic kidney tissues than in matched tumor tissues in 12 matched pairs of freshly frozen primary clear-cell RCC (ccRCC) and nontumor tissue (p=0.002). Immunohistochemical staining showed that CYP4A11 expression was significantly lower in ccRCC than in non-ccRCCs, including papillary, chromophobe, and unclassified RCCs (p<0.001). CYP4A11 expression was associated with PPARα expression, males and high nuclear histologic grades (p=0.001, p=0.018 and p<0.001). Univariate and multivariate analyses revealed that CYP4A11 expression was correlated with short overall survival (p=0.007 and p=0.010).Conclusion: These findings suggest that CYP4A11 expression is a potential poor prognostic factor of RCC. The considerable decrease in CYP4A11 expression is a predictive diagnostic factor of ccRCC, and CYP4A11 metabolism in ccRCC might be different from that in non-ccRCCs.

Prevalence and prognostic value of FBXO11 expression in patients with clear cell renal cell carcinoma

BackgroundFBXO11, a member of the F-box protein family, regulates the cell-cycle by promoting the degradation of Bcl-6 and p53. This protein has been implicated in the progression of several cancers, including renal cell carcinoma (RCC). The aim of this study was to determine the prognostic role of FBXO11 in the clinical outcome of RCC patients.MethodsFBXO11 mRNA expression was analysed in normal and RCC tissue microarrays of the Oncomine database. In addition, the in situ expression levels of stromal FBXO11 protein were assessed in primary RCC tissues from 227 patients (training and validation cohorts) using immunohistochemistry (IHC). Kaplan Meier and Cox regression analyses were used to determine the association between FBXO11 expression and cliniopathological factors. A nomogram was established using the significant prognostic factors to predict overall survival (OS) of RCC patients after one, three and 5 years.ResultsIn the Oncomine database, FBXO11 mRNA levels were lower in normal tissues than in cancer tissues, including clear cell renal cell carcinoma (ccRCC), papillary renal cell carcinoma (pRCC), hereditary ccRCC, non-hereditary ccRCC, VHL mutant ccRCC and VHL wild-type ccRCC. In addition, FBXO11 expression was also significantly higher in metastatic kidney cancer than in primary cancer. Immunohistochemical analysis reported that 57.3% (86 of 150) of the training cohort and 57.1% (44 of 77) of the validation cohort were scored as having high FBXO11 staining density. FBXO11 expression was significantly associated with Fuhrman grade (p = 0.003), UISS score (p = 0.021) and age (p = 0.048) in the training cohort. Furthermore, Kaplan-Meier survival analysis showed that higher FBXO11 levels, T stage, UISS scores and SSIGN score were associated with poor OS in ccRCC patients. Multivariate Cox analysis demonstrated that higher FBXO11 levels and higher UISS score were independent prognostic indicators for OS. Nomogram, calibration plots, AUC values and the C-index showed that the predictive accuracy of conventional prognostic models, including UISS score and SSIGN score, was improved when FBXO11 expression was added.ConclusionsFBXO11 expression was closely related to RCC malignancy and poor prognosis, indicating its potential as a prognostic marker as well as a therapeutic target for RCC.

Value of Ferritin Heavy Chain (FTH1) Expression in Diagnosis and Prognosis of Renal Cell Carcinoma.

BackgroundSerum ferritin is a useful tumor marker for renal cell carcinoma (RCC). However, the expression of ferritin heavy chain (FTH1), the main subunit of ferritin, is unclear in primary RCC tissues. In this study, we investigated FTH1 mRNA expression and its diagnostic and prognostic value in RCC.Material/MethodsThe mRNA expression of FTH1 was analyzed using including Oncomine, Gene Expression Omnibus, and Cancer Genome Atlas datasets, while the protein level of FTH1 was analyzed using the Human Protein Atlas database. The associations between FTH1 and clinicopathologic characteristics and survival time and Cox multivariate survival analysis were analyzed using SPSS 22.0 software. A meta-analysis was performed to assess consistency of FTH1 expression. GO, KEGG, and PPI analyses were used to predict biological functions.ResultsAccording to TCGA data, overexpression of FTH1 was detected in 890 RCC tissues (15.2904±0.63157) compared to 129 normal kidney tissues (14.4502±0.51523, p<0.001). Among the clinicopathological characteristics evaluated, patients with increased pathologic T staging, lymph node metastasis, and distant metastasis were significantly associated with higher expression of FTH1. Elevated FTH1 mRNA levels were correlated with worse prognosis of RCC patients. Cox multivariate survival analysis indicated that age, stage, and M stage were predictors of poor prognosis in patients with RCC.ConclusionsOur data suggest that FTH1 expression is an effective prognostic and diagnosis biomarker for RCC.

HDAC1-induced epigenetic silencing of ASPP2 promotes cell motility, tumour growth and drug resistance in renal cell carcinoma

Renal cell carcinoma (RCC) is highly resistant to chemotherapies. The lack of efficacious treatment for metastatic RCC has led to a poor 5-year survival rate. Here, we found that Apoptosis-stimulating protein of p53-2(ASPP2) was frequently decreased in primary RCC tissues in comparison with non-tumoural kidney controls. Decreased ASPP2 was correlated with high grades and poor outcomes of RCC. Further studies revealed that ASPP2 downregulation promoted EMT and increased resistance to 5-Fluorouracil (5-FU)-induced apoptosis. To this end, the regulatory mechanisms of ASPP2 were further explored. Our data revealed that ASPP2 was inhibited by histone deacetylatlase 1 (HDAC1), which acted by preventing the binding between transcription factor (E2F1) and the ASPP2 promoter. Of particular importance, HDAC1 inhibitor vorinostat restored ASPP2 transcription and produced a synergistic effect with 5-FU in elevating ASPP2, promoting apoptosis and inhibiting EMT in both in vitro and in vivo RCC models. In summary, our data not only highlight an important role of ASPP2 in RCC progression and drug resistance, but also reveal new regulatory mechanisms of ASPP2, which provides important insights into novel treatment strategies by targeting ASPP2 dysregulation in RCC.

A genome-wide comprehensively analyses of long noncoding RNA profiling and metastasis associated lncRNAs in renal cell carcinoma.

Recently, a growing number of studies have indicated that long noncoding RNAs (lncRNAs) are emerging as new critical regulators of tumorigenesis and prognostic markers in multiple cancers. However, the expression pattern of lncRNAs and their contributions in renal cell carcinoma (RCC) remains poorly understood. In this study, we performed a genome-wide comprehensively analysis of lncRNAs profiling and clinical relevance to provide valuable lncRNA candidates for the further study in RCC. RCC and non-tumor tissues RNA sequencing data, and microarray data were obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO), then, these data were annotated and analyzed to find dysregulated lncRNAs in RCC. We identified that hundreds of lncRNAs were differentially expressed in RCC tissues compared with normal tissues, and genomic variation analyses revealed that copy number amplification or deletion happened in some of these lncRNAs genome loci. Moreover, lots of lncRNAs expression levels are significantly associated RCC patients overall survival time, such as PVT1 and DUXAP8. Finally, we identified some novel metastasis associated lncRNAs in RCC (such as DUXAP8) by analyzing lncRNAs profiling in the RCC tissues from patients with metastasis compared with the primary RCC tissues without metastasis; knockdown of DUXAP8 could impair RCC cells invasive ability in vitro. Overall, our findings illuminate a lot of lncRNAs are aberrantly expressed in RCC that may offer useful resource for identification novel prognostic markers in this disease.

Phase III trial of adjuvant sunitinib in patients with high-risk renal cell carcinoma (RCC): Validation of the 16-gene Recurrence Score in stage III patients.

4508 Background: Adjuvant therapy with sunitinib (SU) compared with placebo (PBO) prolonged disease-free survival (DFS) in 615 patients (pts) with high-risk RCC (hazard ratio [HR] 0.76; P= 0.03) in the S-TRAC trial. The 16-gene Recurrence Score (RS) was developed and validated to predict risk of recurrence of RCC after nephrectomy in 2 cohorts of stage I–III pts (Rini et al., Lancet Oncol 2015;16:676-85). We present further validation of RS results in high-risk stage III pts from S-TRAC. Methods: The study was prospectively designed with prespecified genes, algorithm, endpoints, analytical methods, and analysis plan using primary RCC tissues from 212 evaluable pts with informed consent. Gene expression was quantitated by RT-PCR; primary analysis focused on stage III (n = 193 pts). Time to recurrence (TTR) and DFS were analyzed using Cox proportional hazard regression. Results: Baseline characteristics were similar in SU and PBO arms and in pts with and without gene expression data; effect of SU was numerically similar to that in the entire trial (DFS HR 0.78, 95% CI 0.48–1.24; P= 0.29). RS predicted TTR and DFS in both treatment arms with the strongest results observed in PBO arm where high RS group had significantly higher risk (Table). Interaction of RS with treatment was not significant (TTR P= 0.192; DFS P= 0.219); however, the number of events was relatively low. Conclusions: The prognostic value of the 16-gene assay was confirmed in S-TRAC. RS is now validated with consistent results in 2 separate studies (level IB evidence). RS results may help identify patients at high risk who could derive higher absolute benefit from adjuvant treatment. The predictive value of RS to select patients for adjuvant SU requires further investigation in independent adjuvant trials. [Table: see text]

MiR-122 promotes renal cancer cell proliferation by targeting Sprouty2.

MicroRNAs are short non-coding RNAs, which have been implicated in several biological processes. Aberrant expression of the microRNA miR-122 has frequently been reported in malignant cancers. However, the mechanism underlying the effects of miR-122 in renal cell carcinoma remains unknown. The aim of this study was to determine the biological function of miR-122 in renal cell carcinoma and to identify a novel molecular target regulated by miR-122. We measured the expression levels of Sprouty2 in six renal cell carcinoma tissue samples and adjacent non-tumor tissues by western blot analysis. We then used reverse transcription polymerase chain reaction to measure miR-122 levels in 40 primary renal cell carcinoma and adjacent non-malignant tissue samples. The effects of miR-122 down-regulation or Sprouty2 knockdown were evaluated via Cell Counting Kit-8 assay, flow cytometry, and western blot analysis. The relationship between miR-122 and Sprouty2 was determined using dual-luciferase reporter assays. Sprouty2 was down-regulated in renal cell carcinoma tissue samples compared with adjacent normal tissue. In contrast, miR-122 was up-regulated in primary renal cell carcinoma tissue samples compared with adjacent normal tissue samples. Down-regulation of miR-122 substantially weakened the proliferative ability of renal cell carcinoma cell lines in vitro. In contrast, Sprouty2 knockdown promoted the in vitro proliferation of renal cell carcinoma cell lines. The spry2 gene could therefore be a direct target of miR-122. In conclusion, miR-122 could act as a tumor promoter and potentially target Sprouty2. MiR-122 promotes renal cell carcinoma cell proliferation, migration, and invasion and could be a molecular target in novel therapies for renal cell carcinoma.

Differential expression and clinical relevance of MUC1 in renal cell carcinoma metastasis.

To determine the differential expression patterns and prognostic relevance of Mucin-1 (MUC1) expression in clear cell renal cell carcinoma (RCC) metastasis. Tissue microarrays (TMA) from samples of 151 RCC metastases, 61 primary RCCs and corresponding benign renal tissues were immunohistochemically stained for MUC1 and semi-quantitatively evaluated by immunoreactivity scores (IRS). MUC1 differential expression in metastasis, primary RCC and normal tissue were comparatively analyzed. Patient characteristics and clinical follow-up for patients with metastatic RCC (mRCC) were recorded. Correlations of MUC1 expression with mRCC survival were determined. Median cytoplasmic expression was highest in benign tissue (IRS=1.04). Primary RCC (0.50) and metastasis (0.12) showed significantly lower cytoplasmic staining intensity. Membranous expression in benign tissue was, however, significantly lower (0.21) compared with primary RCC (0.59) and metastasis (0.57). Notable differences of MUC1 cytoplasmic and membranous expression were observed between different metastasis sites. Significantly higher (P=0.014) membranous expression was observed in pulmonary versus non-pulmonary lesions, while no significant differences of cytoplasmic MUC1 expression were observed. The prognostic relevance of MUC1 expression in metastatic RCC was limited. MUC1 is differentially expressed in benign renal tissue, primary RCC and RCC metastasis. Membranous MUC1 expression was significantly elevated in pulmonary metastases compared to non-pulmonary lesions, which may reflect individual biology and putative response to MUC1-based anti-cancer therapy.

Abstract 426: Targeting CXCR4 reduced T regulatory cells (Tregs) -mediated cell proliferation suppression in renal cell carcinoma (RCC) patients

Abstract Introduction: Renal cell carcinoma (RCC) is an immunogenic cancer with specimens containing tumor infiltrating lymphocytes. T regulatory cells CD4+CD25+Foxp3+ (Tregs) accumulate in RCC and have been implicated in tumor immune escape. Since the chemokine receptor axis CXCR4/CXCL12/CXCR7 plays a crucial role in renal cancer biology and recent evidences demonstrate that targeting CXCR4 modulate inefficient immunotherapy, the aim of the project was to evaluate patients Tregs function from tumor tissue compared to unaffected surrounding tissue and PBL and to study the effect of CXCR4 antagonism on Tregs immuneresponse. Materials and methods: 26 specimens from RCC, unaffected surrounding tissue and relative PBMCs plus heparinized blood from 15 healthy donors (HD) were collected. Single cells suspensions were obtained; Tregs from renal tumor, unaffected surrounding tissues and peripheral blood and circulating T effector (Teff) cells were isolated using a magnetic isolation cell kit. Treg were identified as CD4/CD25/Foxp3 and characterized for surface activation markers (CTLA-4; CXCR-4; PD-1; CD39; ICOS) by flow cytometry. Tregs suppressive activity was evaluated through in vitro co-colture of purified Tregs versus CFSE-labeled autologous Teff cells. Tregs were added at different ratios and co-cultured for 5 days in presence of anti-CD3/CD28 antibody. CXCR4 antagonism on Tregs suppressive function was evaluated through AMD3100, a selective CXCR4 antagonist, and Peptide R/Peptide R29, a new class of CXCR4 antagonists recently developed (1). Results: Tregs were evaluated in tumor tissue, unaffected surrounding tissue and relative PBMCs from 26 RCC patients and 15 healthy donors (HD). Higher number of Tregs was detected in the RCC tumor tissues and PBMCs compared to HD (p&amp;lt;0.001). Higher expression of activation markers CTLA-4; CXCR4; PD-1; CD39 and ICOS were detected on Treg isolated from tumor specimen compared to PBMCs Tregs cells (p&amp;lt;0.01) and on peripheral Tregs cells compared to HD (p&amp;lt;0.01). Treg suppression of Teff proliferation induced by intratumoral Tregs was more efficient than proliferation suppression induced by circulating and unaffected surrounding tissue isolated Tregs (p&amp;lt;0.05). CXCR4 antagonism through AMD3100, Peptide R and R29 reverted Treg-mediated proliferation suppression (p&amp;lt;0.05). Conclusions: Higher number and suppressive activity was detected for Tregs isolated from primary RCC tissues compared to peritumoral unaffected tissue and PBMCs isolated Tregs. CXCR4 antagonism through AMD3100, Peptide R and R29 prevented T effector cell suppression Tregs induced. Targeting CXCL12/CXCR4 axis in renal cancer might be a strategy for immunotherapeutic intervention. 1. Portella, L., et al., PLoS One, 2013. 8(9): p. e74548 Citation Format: Sara Santagata, Maria Napolitano, Crescenzo D'Alterio, Sabrina Cecere, Renato De Domenico, Carmela Cacciapuoti, Salvatore Dimaro, Luciana Marinelli, Nicola Longo, Sandro Pignata, Sisto Perdonà, Stefania Scala. Targeting CXCR4 reduced T regulatory cells (Tregs) -mediated cell proliferation suppression in renal cell carcinoma (RCC) patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 426. doi:10.1158/1538-7445.AM2015-426

YY1-C/EBPα-miR34a regulatory circuitry is involved in renal cell carcinoma progression

Renal cell carcinoma (RCC) is a common urological malignancy. It remains unclear, however, whether Yin Yang1 (YY1) plays a functional role in the development of human RCC. In the present study, we demonstrated that levels of YY1 were significantly increased in primary RCC tissues when compared to these levels in the matched healthy tissues. YY1 knockdown inhibited cell growth, migration and invasion of RCC cells. Additionally, we highlighted a positive feedback-loop pathway resulting in YY1 upregulation. We observed that overexpression of YY1 caused repression of C/EBPα and the inhibition of C/EBPα led to the suppression of miR-34a. Since YY1 is a direct target of miR-34a, the low level of miR-34a increased the expression of YY1, promoting the aggressiveness of RCC cells. Furthermore, this feedforward mechanism was found in RCC tissues. We observed that miR-34a was downregulated in the pools of cancer tissues when compared to that of the normal tissues. The expression of miR-34a displayed an inverse correlation with YY1, but a positive correlation with C/EBPα. In conclusion, our study highlights the importance of a novel regulatory circuitry for YY1 activation in maintaining the aggressive phenotypes of RCC.

The Expression and Evaluation of Androgen Receptor in Human Renal Cell Carcinoma

To investigate the expression of androgen receptor (AR) with clinical and pathologic features in patients with renal cell carcinoma (RCC) and to explore the function of AR using human RCC cells. The expression of AR was detected by immunohistochemistry in 44 adjacent normal kidney tissues of 120 RCC patients and also in 16 metastatic RCC patients with their respective primary and metastatic tissue samples. The expression of AR was examined by western blot in commonly used human RCC cell lines and normal kidney epithelial cells, and the luciferase assay was performed in those AR-positive RCC cells. The expression rate of AR was higher in adjacent normal kidney (90.9%) than in RCC tissues (30.0%, P <.001), and it was negatively associated with pT stage and Fuhrman's grade. Specifically, there were 40.7% AR-positive cases in pT1 compared with 8.0% in pT3 (P = .013), and 50.0% of grade I cases were found to be AR positive compared with 12.9% in grade III (P = .008). AR expression was slightly higher in primary RCC tissues (12.5%) than their respective metastases (0%, P = .484). AR strongly expressed in CAKI-2 and OSRC-2 cells with little transactivation, which might indicate that AR in those 2 RCC cells has little function. Our results suggest that any attempt to investigate the roles of AR in RCC progression might need to combine the detection of AR expression in tissue samples with examining its function to make a correct correlation between AR and RCC progression.

812P - Role of Genetic Variants in the PI3K/AKT/MTOR Pathway in Renal Cell Carcinoma Patients Treated with Everolimus- A Pilot Study

ABSTRACT Background The phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathway is involved in growth regulation, proliferation control and metabolism in renal cell carcinoma (RCC). In our prospective pilot study, we determined whether common genetic variations in the AKT/ PI3K, FRAP1 (encoding mTOR) are associated with clinical outcomes in RCC patients who underwent palliative therapy with everolimus. Patients and methods Our pilot study was designed to assess everolimus efficacy in RCC patients relapsed after TKI inhibitors (sunitinib and/or sorafenib). Patients were treated with everolimus 10 mg daily orally until disease progression. The genomic DNA was extracted from formalin-fixed, paraffin-embedded primary tumor RCC tissues. 12 potentially functional SNPs in 4 key genes (AKT1, AKT2, FRAP1, PIK3CA) were determined using a real-time PCR genotyping assay and analyzed for association with response to therapy, progression free survival (PFS) and overall survival (OS). Results Median age of 45 enrolled patients was 62 years (range, 41–78). At a median follow-up duration of 8,6 months, the 6-month PFS rate was 53.3%. Partial response was observed in 4,4% (2/45) of the patients. Median PFS was significantly prolonged in the PIK3CA rs6443624 for the CC genotype in comparison to the AA/AC genotype (8.9 months versus 5.5 months, log rank, p= 0.0157). In the multivariate analysis elevated corrected serum calcium and PIK3CA rs6443624 for the AA/AC genotype were unfavorable predictors of PFS (HR 5.25; 95% CI, 1.75–15.75 and HR 3.48; 95%CI, 1.47–8.25, respectively, p Conclusion These results suggest that PIK3CA rs6443624 genetic variation in PI3K/AKT/mTOR pathway may modulate clinical outcomes in patients with RCC who undergo chemotherapy with everolimus. Further clinical studies are needed to confirm these findings. Disclosure All authors have declared no conflicts of interest.

Renal cell carcinoma metastasizing to solitary fibrous tumor of the pleura: a case report

IntroductionA tumor metastasizing to another malignancy is an uncommon phenomenon. Since it was first described in 1902, there have been fewer than 200 cases reported in the literature, with lung cancer metastasizing to renal cell carcinoma being the most frequently described pattern. Here we report a case of a solitary fibrous tumor of the lung acting as the recipient for a renal cell carcinoma. To our knowledge, this is the first reported case of such a combination and the second case involving a solitary fibrous tumor.Case presentationA 58-year-old Caucasian man who developed a persistent dry cough presented to our hospital. Imaging studies revealed a large pleural-based mass in the left lung. A biopsy of the mass showed a spindle-cell lesion consistent with a solitary fibrous tumor. The patient underwent surgical excision of the 13 cm mass. The pathological examination confirmed the diagnosis of a solitary fibrous tumor but also demonstrated discrete foci of metastatic renal cell carcinoma. Until that point, a primary renal cell carcinoma tissue diagnosis had not been made and the initial radiological work-up was inconclusive.ConclusionAwareness of the unusual phenomenon of tumor-to-tumor metastasis is important for practicing surgical pathologists, particularly in the evaluation of a mass lesion showing bimodal histology. This case also highlights the importance of careful examination of surgical specimens, as minute and unusual findings can direct patient care.

Wnt antagonist DKK1 acts as a tumor suppressor gene that induces apoptosis and inhibits proliferation in human renal cell carcinoma

The functional significance of Wnt antagonist DKK1 has not been investigated in renal cell carcinoma (RCC). Therefore, we hypothesized that DKK1 may be a tumor suppressor gene and is epigenetically silenced, thus decreased DKK1 may cause progression of RCC. To assess the function of DKK1, we established stable DKK1 transfected cells and monitored them regarding cell viability, colony formation, apoptosis, cell cycle, and invasive capability. RCC cell lines had decreased levels of DKK1, which were increased after treatment with 5-Aza-2'-deoxycytidine and trichostatin A. In chromatin immunoprecipitation assay, the level of dimethyl H3K9 and trimethyl H3K27 was decreased after 5-Aza-2'-deoxycytidine/trichostatin A treatment in RCC cell lines. Increased methylation was also associated with higher pathological stages in primary RCC tissues. T-cell factor/lymphoid enhancer factor activity and nuclear beta-catenin expression were not changed in DKK1 transfectants. Also the expression of cyclinD1 and c-Myc was not changed in DKK1 transfectants. These results suggest that DKK1 may not be involved in the beta-catenin dependent pathway. We also evaluated the expression of various related genes. Cleaved caspase3, p53, p21 and puma expression were significantly upregulated in the DKK1 transfected cells. The population of apoptotic cells was increased in stable DKK1 cells and tumor growth suppression was also observed in nude mice with DKK1 transfected cells. In conclusion, this is the first report to show that DKK1 expression is epigenetically silenced in kidney cancer and its reexpression induces apoptosis and cell cycle arrest in RCC.

Identification of an Immunogenic CTL Epitope of HIFPH3 for Immunotherapy of Renal Cell Carcinoma

CD8(+) CTLs have an essential role in immune response against tumor. Although tumor-associated antigens have been identified in renal cell carcinoma (RCC), few of these are commonly shared and investigated as therapeutic targets in the clinical medicine. In this report, we show that HIFPH3, a member of prolyl hydroxylases that function as oxygen sensor, is a novel tumor antigen and HIFPH3-specific CTLs are induced from peripheral blood lymphocytes of RCC patients. Expression of HIFPH3 was examined by reverse transcription-PCR and immunostaining with anti-HIFPH3 antibody. To identify HLA-A24-restricted T-cell epitopes of HIFPH3, eight peptides were selected from the amino acid sequence of this protein and screened for their binding affinity to HLA-A24. Peptide-specific CTLs were induced by stimulating peripheral blood lymphocytes of HLA-A24-positive RCC patients with these peptides in vitro. HLA-A24-restricted cytotoxicity of the CTLs against HIFPH3(+) RCC lines was assessed by chromium release assay. HIFPH3 was overexpressed in many RCC cell lines and primary RCC tissues, whereas it was not detectable in normal adult tissues by reverse transcription-PCR. Of the eight peptides that contained HLA-A24-binding motif, HIFPH3-8 peptide (amino acid sequence, RYAMTVWYF) could induce the peptide-specific CTLs from 3 of 6 patients with HIFPH3-positive RCC. Furthermore, HIFPH3-8 peptide-specific CTLs showed cytotoxicity against HIFPH3(+) RCC cell lines in a HLA-A24-restricted manner. HIFPH3 may be a target antigen in immunotherapy for RCC and HIFPH3-8 peptide could be used as a peptide vaccine for HLA-A*2402(+)/HIFPH3(+) RCC patients.

Macrophage Inflammatory Protein–1δ: A Novel Osteoclast Stimulating Factor Secreted by Renal Cell Carcinoma Bone Metastasis

Approximately 30% of patients with renal cell carcinoma (RCC) develop bone metastasis, which is characterized by extensive osteolysis leading to severe bone pain and pathologic fracture. Although the mechanism of RCC-induced osteolysis is unknown, studies of bone metastasis have shown that tumor-induced changes in bone remodeling are likely mediated by alterations in the bone microenvironment. Here, we report the discovery of a novel osteoclast stimulatory factor secreted by RCC bone metastasis (RBM). Through microarray analysis, we found expression of the chemokine, macrophage inflammatory protein-1 delta (MIP-1 delta), to be increased in RBM versus patient-matched primary RCC tissues and confirmed this finding by quantitative reverse transcription-PCR (qRT-PCR) and ELISA (P < 0.05). Furthermore, MIP-1 delta expression in RBM tissues was significantly (P < 0.001) higher than in human bone marrow, suggesting a potential alteration of the bone microenvironment. The receptors for MIP-1 delta, CCR1 and CCR3, were expressed in both osteoclast precursors and mature, bone-resorbing osteoclasts as shown by qRT-PCR and Western analysis. In functional studies, MIP-1 delta stimulated chemotaxis of two osteoclast precursor cell types: murine bone marrow mononuclear cells (BM-MNC) and RAW 264.7 cells. Furthermore, MIP-1 delta treatment of murine calvaria caused increased bone resorption as determined by measurement of released calcium. Correspondingly, MIP-1 delta significantly enhanced osteoclast formation and activity in response to RANKL in both BM-MNC and RAW 264.7 cells. Taken together, these data suggest that MIP-1 delta expression is increased in RBM relative to RCC and bone marrow, and may promote RBM-induced osteolysis by stimulating the recruitment and differentiation of osteoclast precursors into mature osteoclasts.

Cathepsin D Serum Levels Are Not a Valid Serum Marker in Renal Cell Carcinoma

Background: It was the aim of this study to evaluate the serum levels of cathepsin D (Cath D) as a diagnostic tool in patients with renal cell carcinoma (RCC) in comparison with healthy volunteers. Methods: Cath D serum levels were measured in serum samples obtained preoperatively from 32 patients with histologically confirmed RCC versus 30 healthy individuals using an enzyme immunoassay. Additionally, for the tumor group, Cath D serum levels were correlated with tumor stage and grade as determined according to the 2002 TNM classification. Results: The serum Cath D concentration was not significantly different in patients with RCC compared with healthy individuals (mean 16.58 vs. 16.64 ng/ml; p = 0.43). Furthermore, there was no significant association between Cath D serum levels and several patient or tumor characteristics such as tumor stage, tumor grade, lymph node status, presence of metastasis, gender or age. Conclusions: In contrast to overexpression of Cath D in primary RCC tissue, serum Cath D is not altered in RCC patients when compared with healthy volunteers. In this small cohort, Cath D serum levels did not reveal additional clinical information in patients diagnosed with a small renal mass. Further prospective multicenter studies might shed more light on the value of Cath D in the diagnostics of RCC.

Imprinted DLK1 is a putative tumor suppressor gene and inactivated by epimutation at the region upstream of GTL2 in human renal cell carcinoma

A common deletion at chromosomal arm 14q32 in human renal cell carcinoma (RCC) prompted us to explore a tumor suppressor gene (TSG) in this region. We report that imprinted DLK1 at 14q32, a regulator of adipocyte differentiation, is a candidate TSG in RCCs. DLK1 expression was lost in 39 out of 50 (78%) primary RCC tissues, whereas expression of DLK1 was maintained in every normal kidney tissue examined. DLK1 was expressed in only one of 15 (7%) RCC-derived cell lines. In order to see the biological significance of DLK1 inactivation in RCCs, we tested the effect of restoration of DLK1 in RCC cell lines, using a recombinant retrovirus containing the gene. Reintroduction of DLK1 into DLK1-null RCC cell lines markedly increased anchorage-independent cell death, anoikis and suppressed tumor growth in nude mice. We then investigated the underlying mechanisms for DLK1 inactivation in RCCs. We found loss of heterozygosity at this region in 12 out of 50 RCC tissues (24%). To explore the role of epigenetic regulation of DLK1 inactivation in RCCs, we conducted methylation analysis of the upstream region and the gene body of DLK1. We could not find a differentially methylated region in either the upstream region or the gene body of DLK1. However, we found that gain of methylation upstream of GTL2, a reciprocal imprinted gene for DLK1, is a critical epigenetic alteration for the inactivation of DLK1 in RCCs. The present data have shown that gain of methylation upstream of the untranslated GTL2 leads to pathological downregulation of DLK1 in RCCs.