Zhongfa Zhang

Inhibition of MAPK kinase signaling pathways suppressed renal cell carcinoma growth and angiogenesis in vivo.

The mitogen-activated protein kinase (MAPK) signaling pathways play essential roles in cell proliferation and differentiation. Recent studies also show the activation of MAPK signaling pathways in tumorigenesis, metastasis, and angiogenesis of multiple human malignancies, including renal cell carcinoma (RCC). To assess the role of this pathway in regulating the proliferation and survival of RCC cells, we first examined the expression of MAPK kinase (MKK) and MAPK in clear cell RCC and confirmed the overexpression of MKK1 and extracellular signal-regulated kinase 2 (ERK2) in these tumors. We then tested the effects of pharmacologic inhibition of MKK on human RCC cell lines, both in vitro and in vivo, using anthrax lethal toxin (LeTx), which cleaves and inactivates several MKKs. Western blotting showed that the phosphorylation levels of ERK, c-Jun-NH(2) kinase, and p38 MAPK decreased after 72 h of LeTx treatment. Exposure to LeTx for 72 h reduced cell proliferation by 20% without significant effects on cell cycle distribution and apoptosis. Anchorage-independent growth of RCC cells was dramatically inhibited by LeTx. In vivo studies showed that tumor growth of RCC xenografts could be suppressed by LeTx. Extensive necrosis and decreased tumor neovascularization were observed after LeTx treatment. LeTx also showed direct inhibition of proliferation of endothelial cells in vitro. Our results suggest that suppression of one or more MAPK signaling pathways may inhibit RCC growth through the disruption of tumor vasculature.

Parafibromin, a Component of the Human PAF Complex, Regulates Growth Factors and Is Required for Embryonic Development and Survival in Adult Mice

ABSTRACT Parafibromin, a transcription factor associated with the PAF complex, is encoded by the HRPT2 gene, mutations of which cause the hyperparathyroidism-jaw tumor syndrome (OMIM145001). To elucidate the function of parafibromin, we generated conventional and conditional Hrpt2 knockout mice and found that Hrpt2−/− mice were embryonic lethal by embryonic day 6.5 (E6.5). Controlled deletion of Hrpt2 after E8.5 resulted in apoptosis and growth retardation. Deletion of Hrpt2 in adult mice led to severe cachexia and death within 20 days. To explore the mechanism underlying the embryonic lethality and death of adult mice, mouse embryonic fibroblasts (MEFs) were cultured and Hrpt2 was deleted in vitro. Hrpt2−/− MEFs underwent apoptosis, while Hrpt2+/+ and Hrpt2+/− MEFs grew normally. To study the mechanism of this apoptosis, Hrpt2+/+ and Hrpt2−/− MEFs were used in cDNA microarray, semiquantitative reverse transcription-PCR, and chromatin immunoprecipitation assays to identify genes regulated by parafibromin. These revealed that Hrpt2 expression and the parafibromin/PAF complex directly regulate genes involved in cell growth and survival, including H19, Igf1, Igf2, Igfbp4, Hmga1, Hmga2, and Hmgcs2. Thus, our results show that expression of Hrpt2 and parafibromin is pivotal in mammalian development and survival in adults and that these functions are likely mediated by the transcriptional regulation of growth factors.

Serglycin Is a Theranostic Target in Nasopharyngeal Carcinoma that Promotes Metastasis

Nasopharyngeal carcinoma (NPC) is known for its high-metastatic potential. Here we report the identification of the proteoglycan serglycin as a functionally significant regulator of metastasis in this setting. Comparative genomic expression profiling of NPC cell line clones with high- and low-metastatic potential revealed the serglycin gene (SRGN) as one of the most upregulated genes in highly metastatic cells. RNAi-mediated inhibition of serglycin expression blocked serglycin secretion and the invasive motility of highly metastatic cells, reducing metastatic capacity in vivo. Conversely, serglycin overexpression in poorly metastatic cells increased their motile behavior and metastatic capacity in vivo. Growth rate was not influenced by serglycin in either highly or poorly metastatic cells. Secreted but not bacterial recombinant serglycin promoted motile behavior, suggesting a critical role for glycosylation in serglycin activity. Serglycin inhibition was associated with reduced expression of vimentin but not other epithelial-mesenchymal transition proteins. In clinical specimens, serglycin expression was elevated significantly in liver metastases from NPC relative to primary NPC tumors. We evaluated the prognostic value of serglycin by immunohistochemical staining of tissue microarrays from 263 NPC patients followed by multivariate analyses. High serglycin expression in primary NPC was found to be an unfavorable independent indicator of distant metastasis-free and disease-free survival. Our findings establish that glycosylated serglycin regulates NPC metastasis via autocrine and paracrine routes, and that it serves as an independent prognostic indicator of metastasis-free survival and disease-free survival in NPC patients.

Somatic Pairing of Chromosome 19 in Renal Oncocytoma Is Associated with Deregulated ELGN2-Mediated Oxygen-Sensing Response

Chromosomal abnormalities, such as structural and numerical abnormalities, are a common occurrence in cancer. The close association of homologous chromosomes during interphase, a phenomenon termed somatic chromosome pairing, has been observed in cancerous cells, but the functional consequences of somatic pairing have not been established. Gene expression profiling studies revealed that somatic pairing of chromosome 19 is a recurrent chromosomal abnormality in renal oncocytoma, a neoplasia of the adult kidney. Somatic pairing was associated with significant disruption of gene expression within the paired regions and resulted in the deregulation of the prolyl-hydroxylase ELGN2, a key protein that regulates the oxygen-dependent degradation of hypoxia-inducible factor (HIF). Overexpression of ELGN2 in renal oncocytoma increased ubiquitin-mediated destruction of HIF and concomitantly suppressed the expression of several HIF-target genes, including the pro-death BNIP3L gene. The transcriptional changes that are associated with somatic pairing of chromosome 19 mimic the transcriptional changes that occur following DNA amplification. Therefore, in addition to numerical and structural chromosomal abnormalities, alterations in chromosomal spatial dynamics should be considered as genomic events that are associated with tumorigenesis. The identification of EGLN2 as a significantly deregulated gene that maps within the paired chromosome region directly implicates defects in the oxygen-sensing network to the biology of renal oncocytoma.

The Investigational Aurora Kinase A Inhibitor MLN8237 Induces Defects in Cell Viability and Cell-Cycle Progression in Malignant Bladder Cancer Cells In Vitro and In Vivo

Purpose: Despite more than 70,000 new cases of bladder cancer in the United States annually, patients with advanced disease have a poor prognosis due to limited treatment modalities. We evaluated Aurora kinase A, identified as an upregulated candidate molecule in bladder cancer, as a potential therapeutic target. Experimental Design: Gene expression in human bladder cancer samples was evaluated using RNA microarray and quantitative reverse transcriptase PCR. Effects of the Aurora kinase A inhibitor MLN8237 (Millennium) on cell dynamics in malignant T24 and UM-UC-3 and papilloma-derived RT4 bladder cells were evaluated in vitro and in vivo in a mouse xenograft model. Results: A set of 13 genes involved in the mitotic spindle checkpoint, including Aurora kinases A and B, were upregulated in human urothelial carcinoma compared with normal urothelium. The Aurora kinase A inhibitor MLN8237 induced cell-cycle arrest, aneuploidy, mitotic spindle failure, and apoptosis in the human bladder cancer cell lines T24 and UM-UC-3. MLN8237 also arrested tumor growth when administered orally over 4 weeks in a mouse bladder cancer xenograft model. Finally, in vitro sequential administration of MLN8237 with either paclitaxel or gemcitabine resulted in synergistic cytotoxic effects in T24 cells. Conclusions: Mitotic spindle checkpoint dysfunction is a common characteristic of human urothelial carcinoma and can be exploited with pharmacologic Aurora A inhibition. Given our demonstration of the ability of the Aurora A inhibitor MLN8237 to inhibit growth of bladder cancer in vitro and in vivo, we conclude that Aurora kinase inhibitors warrant further therapeutic investigation in bladder cancer. Clin Cancer Res; 19(7); 1717–28. ©2013 AACR.

Radiosensitization by inhibiting STAT1 in renal cell carcinoma.

PURPOSE Renal cell carcinoma (RCC) has been historically regarded as a radioresistant malignancy, but the molecular mechanism underlying its radioresistance is not understood. This study investigated the role of signal transducer and activator of transcription 1 (STAT1), a transcription factor downstream of the interferon-signaling pathway, in radioresistant RCC. METHODS AND MATERIALS The expressions of STAT1 and STAT3 in 164 human clear cell RCC samples, 47 papillary RCC samples, and 15 normal kidney tissue samples were examined by microarray expression profiling and immunohistochemistry. Western blotting was performed to evaluate the total and phosphorylated STAT1 expression in CRL-1932 (786-O) (human clear cell RCC), SKRC-39 (human papillary RCC), CCL-116 (human fibroblast), and CRL-1441 (G-401) (human Wilms tumor). STAT1 was reduced or inhibited by fludarabine and siRNA, respectively, and the effects on radiation-induced cell death were investigated using clonogenic assays. RESULTS STAT1 expression, but not STAT3 expression, was significantly greater in human RCC samples (p = 1.5 x 10(-8) for clear cell; and p = 3.6 x 10(-4) for papillary). Similarly, the expression of STAT1 was relatively greater in the two RCC cell lines. STAT1 expression was reduced by both fludarabine and siRNA, significantly increasing the radiosensitivity in both RCC cell lines. CONCLUSION This is the first study reporting the overexpression of STAT1 in human clear cell and papillary RCC tissues. Radiosensitization in RCC cell lines was observed by a reduction or inhibition of STAT1 signaling, using fludarabine or siRNA. Our data suggest that STAT1 may play a key role in RCC radioresistance and manipulation of this pathway may enhance the efficacy of radiotherapy.

Comparative gene expression profiling analysis of urothelial carcinoma of the renal pelvis and bladder

BackgroundUrothelial carcinoma (UC) can arise at any location along the urothelial tract, including the urethra, bladder, ureter, or renal pelvis. Although tumors arising in these various locations have similar morphology, it is unclear whether the gene expression profiles are similar between the upper-tract (ureter and renal pelvis) and lower-tract (bladder and urethra) carcinomas. Because differences may facilitate different screening and treatment modalities, we sought to examine the relationship between urothelial carcinoma of the renal pelvis (rUC) and urothelial carcinoma of the bladder (bUC).MethodsFresh tumor tissue was collected from patients with bUC (n = 10) and benign mucosa from the bladder of individuals undergoing resection for non-UC conditions (n = 7). Gene expression profiles from these samples were determined using high-throughput Affymetrix gene expression microarray chips. Bioinformatic approaches were used to compare the gene expression profiles of these samples with those of rUC samples and normal kidney samples that had been described previously.ResultsUsing unsupervised analytic approaches, rUC and bUC were indistinguishable. Yet when a supervised analytic approach was used, a small number of differentially expressed genes were identified; these differences were most likely limited to a single pathway--the chloride ion binding activity pathway--which was more frequently activated in rUC than in bUC.ConclusionsWe found that the gene expression profiles of UCs from the upper and lower tract were extremely similar, suggesting that similar pathogenic mechanisms likely function in the development of these tumors. The differential expression of genes in the identified pathway may represent a new avenue for detection of upper-tract tumors.

Detection of submicroscopic constitutional chromosome aberrations in clinical diagnostics: a validation of the practical performance of different array platforms

For several decades etiological diagnosis of patients with idiopathic mental retardation (MR) and multiple congenital anomalies (MCA) has relied on chromosome analysis by karyotyping. Conventional karyotyping allows a genome-wide detection of chromosomal abnormalities but has a limited resolution. Recently, array-based comparative genomic hybridization (array CGH) technologies have been developed to evaluate DNA copy-number alterations across the whole-genome at a much higher resolution. It has proven to be an effective tool for detection of submicroscopic chromosome abnormalities causing congenital disorders and has recently been adopted for clinical applications. Here, we investigated four high-density array platforms with a theoretical resolution ⩽100 kb: 33K tiling path BAC array, 500K Affymetrix SNP array, 385K NimbleGen oligonucleotide array and 244K Agilent oligonucleotide array for their robustness and implementation in our diagnostic setting. We evaluated the practical performance based on the detection of 10 previously characterized abnormalities whose size ranged from 100 kb to 3 Mb. Furthermore, array data analysis was performed using four computer programs developed for each corresponding platform to test their effective ability of reliable copy-number detection and their user-friendliness. All tested platforms provided sensitive performances, but our experience showed that accurate and user-friendly computer programs are of crucial importance for reliable copy-number detection.

Small-molecule inhibitors targeting the DNA-binding domain of STAT3 suppress tumor growth, metastasis and STAT3 target gene expression in vivo.

Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in malignant tumors and has important roles in multiple aspects of cancer aggressiveness. Thus targeting STAT3 promises to be an attractive strategy for treatment of advanced metastatic tumors. Although many STAT3 inhibitors targeting the SH2 domain have been reported, few have moved into clinical trials. Targeting the DNA-binding domain (DBD) of STAT3, however, has been avoided due to its ‘undruggable‘ nature and potentially limited selectivity. In a previous study, we reported an improved in silico approach targeting the DBD of STAT3 that resulted in a small-molecule STAT3 inhibitor (inS3-54). Further studies, however, showed that inS3-54 has off-target effect although it is selective to STAT3 over STAT1. In this study, we describe an extensive structure and activity-guided hit optimization and mechanistic characterization effort, which led to identification of an improved lead compound (inS3-54A18) with increased specificity and pharmacological properties. InS3-54A18 not only binds directly to the DBD and inhibits the DNA-binding activity of STAT3 both in vitro and in situ but also effectively inhibits the constitutive and interleukin-6-stimulated expression of STAT3 downstream target genes. InS3-54A18 is completely soluble in an oral formulation and effectively inhibits lung xenograft tumor growth and metastasis with little adverse effect on animals. Thus inS3-54A18 may serve as a potential candidate for further development as anticancer therapeutics targeting the DBD of human STAT3 and DBD of transcription factors may not be ‘undruggable‘ as previously thought.

Expression of the PTTG1 oncogene is associated with aggressive clear cell renal cell carcinoma

The pituitary tumor transforming gene (PTTG1) is a recently discovered oncogene implicated in malignant progression of both endocrine and nonendocrine malignancies. Clear cell renal cell carcinoma (ccRCC) is cytogenetically characterized by chromosome 3p deletions that harbor the ccRCC-related von Hippel-Lindau, PBRM1, BAP1, and SETD2 tumor suppressor genes, along with chromosome 5q amplifications where the significance has been unclear. PTTG1 localizes to the chromosome 5q region where amplifications occur in ccRCC. In this study, we report a functional role for PTTG1 in ccRCC tumorigenesis. PTTG1 was amplified in ccRCC, overexpressed in tumor tissue, and associated with high-grade tumor cells and poor patient prognosis. In preclinical models, PTTG1 ablation reduced tumorigenesis and invasion. An analysis of gene expression affected by PTTG1 indicated an association with invasive and metastatic disease. PTTG1-dependent expression of the RhoGEF proto-oncogene ECT2 was observed in a number of ccRCC cell lines. Moreover, ECT2 expression correlated with PTTG1 expression and poor clinical features. Together, our findings reveal features of PTTG1 that are consistent with its identification of an oncogene amplified on chromsome 5q in ccRCC, where it may offer a novel therapeutic target of pathologic significance in this disease.