Identification and characterization of cellular genes differentially expressed in a subset of tumors with non -functional WT1

Wilms tumor (WT) or nephroblastoma is a genetically heterogeneous pediatric renal tumor that accounts for 6–7% of all childhood cancers in the U.S. WT1, located at 11p13, is the sole WT gene cloned to date. Additional genomic regions containing genes that play a role in the development of Wilms tumor include 11p15, 7p, 16q, 1p, 17q and 19q. This heterogeneity has made it extremely difficult to develop an understanding of the pathways involved in the development of WT, even in the 5–20% of tumors that show mutations at the WT1 locus. My research addresses this gap in our current comprehension of the development of WT. ^ I have used two complementary approaches to extend the current understanding of molecular changes involved in the development of WT. In order to minimize complexities due to genetic heterogeneity, I confined my analysis to the WT1 pathway by assessing those genetically defined tumors that carry WT1 mutations. WT1 encodes a zinc finger transcription factor, and in vitro studies have identified many genes that are potentially regulated in vivo by WT1. However, there is very little in vivo data that suggests that they are transcriptionally regulated endogenously by WT1. In one approach I assessed the role of WT1 in the in vivo regulation of PDGFA and IGF2, two genes that are strong contenders for endogenous regulation by WT1. Using primary tissue samples, I found no correlation between the level of RNA expression of WT1 with either PDGFA or IGF2, suggesting that WT1 does not play a critical role in their expression in either normal kidney or WT. ^ In a parallel strategy, using differential display analysis I compared global gene expression in a subset of tumors with known homozygous inactivating WT1 mutations (WT1-tumors) to the gene expression in a panel of appropriate control tissues (fetal kidney, normal kidney, rhabdoid tumor and pediatric renal cell carcinoma). Transcripts that are aberrantly expressed in this subset of Wilms tumors are candidates for endogenous transcriptional regulation by WT1 as well as for potentially functioning in the development of WT. By this approach I identified several differentially expressed transcripts. I further characterized two of these transcripts, identifying a candidate WT gene in the process. I then performed a detailed analysis of this WT candidate gene, which maps to 7p. Future studies will shed more light on the role of these differentially expressed genes in WT. ^

Defective placental vasculogenesis causes embryonic lethality in VHL-deficient mice

Inheritance of an inactivated form of the VHL tumor suppressor gene predisposes patients to develop von Hippel–Lindau disease, and somatic VHL inactivation is an early genetic event leading to the development of sporadic renal cell carcinoma. The VHL gene was disrupted by targeted homologous recombination in murine embryonic stem cells, and a mouse line containing an inactivated VHL allele was generated. While heterozygous VHL (+/−) mice appeared phenotypically normal, VHL −/− mice died in utero at 10.5 to 12.5 days of gestation (E10.5 to E12.5). Homozygous VHL −/− embryos appeared to develop normally until E9.5 to E10.5, when placental dysgenesis developed. Embryonic vasculogenesis of the placenta failed to occur in VHL −/− mice, and hemorrhagic lesions developed in the placenta. Subsequent hemorrhage in VHL −/− embryos caused necrosis and death. These results indicate that VHL expression is critical for normal extraembryonic vascular development.

Elongin BC complex prevents degradation of von Hippel-Lindau tumor suppressor gene products

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene causes the familial cancer syndrome, VHL disease, characterized by a predisposition to renal cell carcinoma and other tumor types. Loss of VHL gene function also is found in a majority of sporadic renal carcinomas. A preponderance of the tumor-disposing inherited missense mutations detected in VHL disease are within the elongin-binding domain of VHL. This region mediates the formation of a multiprotein VHL complex containing elongin B, elongin C, cul-2, and Rbx1. This VHL complex is thought to function as an E3 ubiquitin ligase. Here, we report that VHL proteins harboring mutations which disrupt elongin binding are unstable and rapidly degraded by the proteasome. In contrast, wild-type VHL proteins are directly stabilized by associating with both elongins B and C. In addition, elongins B and C are stabilized through their interactions with each other and VHL. Thus, the entire VHL/elongin complex is resistant to proteasomal degradation. Because the elongin-binding domain of VHL is frequently mutated in cancers, these results suggest that loss of elongin binding causes tumorigenesis by compromising VHL protein stability and/or potential VHL ubiquitination functions.

Angiogenesis in cancer - general pathways and their therapeutic implications

A vast amount of data shows that angiogenesis has a pivotal role in tumor growth, progression, invasiveness and metastasis. This is a complex process involving essential signaling pathways such as vascular endothelial growth factor (VEGF) and Notch in vasculature, as well as additional players such as bone marrow-derived endothelial progenitor cells. Primary tumor cells, stromal cells and cancer stem cells strongly influence vessel growth in tumors. Better understanding of the role of the different pathways and the crosstalk between different cells during tumor angiogenesis are crucial factors for developing more effective anticancer therapies. Targeting angiogenic factors from the VEGF family has become an effective strategy to inhibit tumor growth and so far the most successful results are seen in metastatic colorectal cancer (CRC), renal cell carcinoma (RCC) and non-small cell lung cancer (NSCLL). Despite the initial enthusiasm, the angiogenesis inhibitors showed only moderate survival benefit as monotherapy, along with a high cost and many side effects. Obviously, other important pathways may affect the angiogenic switch, among them Notch signaling pathway attracted a large interest because its ubiquitous role in carcinogenesis and angiogenesis. Herein we present the basics for VEGF and Notch signaling pathways and current advances of targeting them in antiangiogenic, antitumor therapy.

Haemodynamic assessment following inferior vena cava resection without replacement

Background: Treatment of bulky retroperitoneal malignancy may require en bloc resection of the infrarenal inferior vena cava. A number of reconstructive options are available to the surgeon but objective haemodynamic assessment of the peripheral venous system following resection without replacement is lacking. The aim of the present paper was thus to determine the symptomatic and haemodynamic effects of not reconstructing the resected infrarenal inferior vena cava. Methods: A retrospective descriptive study was carried out at Princess Alexandra Hospital in Queensland. Five patients underwent resection of the thrombosed infrarenal inferior vena cava as part of retroperitoneal lymph node dissection for testicular cancer (n = 3), radical nephrectomy for renal cell carcinoma (n = 1) and thrombosed inferior vena cava aneurysm (n = 1). Clinical effects were determined via the modified venous clinical severity score and venous disability score. Haemodynamic data were obtained postoperatively using venous duplex ultrasound and air plethysmography. Results: None of the present patients scored >2 (out of 30) on the modified venous clinical severity score or >1 (out of 3) on the venous disability score. Haemodynamic studies showed only minor abnormalities. Conclusions: Not reconstructing the resected thrombosed infrarenal inferior vena cava results in minor signs and symptoms of peripheral venous hypertension and only minor abnormalities on haemodynamic assessment.

Modulation of human V alpha 24(+)V beta 11(+) NKT cells by age, malignancy and conventional anticancer therapies

Immunotherapy strategies aimed at increasing human Valpha24(+)Vbeta11(+) natural killer T (NKT) cell numbers are currently a major focus. To provide further information towards the goal of NKT cell-based immunotherapy, we assessed the effects of age, cancer status and prior anticancer treatment on NKT cell numbers and their expansion capacity following alpha-galactosylceramide (alpha-GalCer) stimulation. The percentage and absolute number of peripheral blood NKT cells was assessed in 40 healthy donors and 109 solid cancer patients ( colorectal ( n = 33), breast ( n = 10), melanoma ( n = 17), lung ( n = 8), renal cell carcinoma ( n = 10), other cancers ( n = 31)). Responsiveness to alpha-GalCer stimulation was also assessed in 28 of the cancer patients and 37 of the healthy donors. Natural killer T cell numbers were significantly reduced in melanoma and breast cancer patients. While NKT numbers decreased with age in healthy donors, NKT cells were decreased in these cancer subgroups despite age and sex adjustments. Prior radiation treatment was shown to contribute to the observed reduction in melanoma patients. Although cancer patient NKT cells were significantly less responsive to alpha-GalCer stimulation, they remained capable of substantial expansion. Natural killer T cells are therefore modulated by age, malignancy and prior anticancer treatment; however, cancer patient NKT cells remain capable of responding to alpha-GalCer-based immenotherapies.

Protein kinase C activation and its role in kidney disease

Protein kinase C (PKC) comprises a superfamily of isoenzymes, many of which are activated by cofactors such as diacylglycerol and phosphatidylserine. In order to be capable of activation, PKC must first undergo a series of phosphorylations. In turn, activated PKC phosphorylates a wide variety of intracellular target proteins and has multiple functions in signal transduced cellular regulation. A role for PKC activation had been noted in several renal diseases, but two that have had most investigation are diabetic nephropathy and kidney cancer. In diabetic nephropathy, an elevation in diacylglycerol and/or other cofactor stimulants leads to an increase in activity of certain PKC isoforms, changes that are linked to the development of dysfunctional vasculature. The ability of isoform-specific PKC inhibitors to antagonize diabetes-induced vascular disease is a new avenue for treatment of this disorder. In the development and progressive invasiveness of kidney cancer, increased activity of several specific isoforms of PKC has been noted. It is thought that this may promote the kidney cancer's inherent resistance to apoptosis, in natural regression or after treatments, or it may promote the invasiveness of renal cancers via cellular differentiation pathways. In general, however, a more complete understanding of the functions of individual PKC isoforms in the kidney, and development or recognition of specific inhibitors or promoters of their activation, will be necessary to apply this knowledge for treatment of cellular dysregulation in renal disease.

Aptamers: a promising chemical antibody for cancer therapy

Aptamers, also known as chemical antibodies, are single-stranded nucleic acid oligonucleotides which bind to their targets with high specificity and affinity. They are typically selected by repetitive in vitro process termed systematic evolution of ligands by exponential enrichment (SELEX). Owing to their excellent properties compared to conventional antibodies, notably their smaller physical size and lower immunogenicity and toxicity, aptamers have recently emerged as a new class of agents to deliver therapeutic drugs to cancer cells by targeting specific cancer-associated hallmarks. Aptamers can also be structurally modified to make them more flexible in order to conjugate other agents such as nano-materials and therapeutic RNA agents, thus extending their applications for cancer therapy. This review presents the current knowledge on the practical applications of aptamers in the treatment of a variety of cancers.

Hypertension, antihypertensive treatment and cancer incidence and mortality : a pooled collaborative analysis of 12 Australian and New Zealand cohorts

BACKGROUND: Observational studies examining associations between hypertension and cancer are inconsistent. We explored the association of hypertension, graded hypertension and antihypertensive treatment with cancer incidence and mortality. METHOD: Eighty-six thousand five hundred and ninety-three participants from the Australian and New Zealand Diabetes and Cancer Collaboration were linked to the National Death Index and Australian Cancer Database. Cox proportional hazards models estimated hazard ratios and 95% confidence intervals (95% CI) for the association of treated and untreated hypertension with cancer incidence and mortality. RESULTS: Over a median follow-up of 15.1 years, 12 070 incident and 4350 fatal cancers were identified. Untreated and treated hypertension, compared with normotension, were associated with an increased risk for cancer incidence [hazard ratio 1.06, 95% CI (1.00-1.11) and 1.09 (1.02-1.16) respectively], and cancer mortality (1.07, 0.98-1.18) and (1.15, 1.03-1.28), respectively. When compared with untreated hypertension, treated hypertension did not have a significantly greater risk for cancer incidence (1.03, 0.97-1.10) or mortality (1.07, 0.97-1.19). A significant dose-response relationship was observed between graded hypertension and cancer incidence and mortality; Ptrend = 0.053 and Ptrend = 0.001, respectively. When stratified by treatment status, these relationships remained significant in untreated, but not in treated, hypertension. CONCLUSION: Hypertension, both treated and untreated, is associated with a modest increased risk for cancer incidence and mortality. Similar risks in treated and untreated hypertension suggest that the increased cancer risk is not explained by the use of antihypertensive treatment.

Phase III trial of gemcitabine and carboplatin versus mitomycin, ifosfamide, and cisplatin or mitomycin, vinblastine, and cisplatin in patients with advanced nonsmall cell lung carcinoma

BACKGROUND. The authors compared gemcitabine and carboplatin (GC) with mitomycin, ifosfamide, and cisplatin (MIC) or mitomycin, vinblastine, and cisplatin (MVP) in patients with advanced nonsmall cell lung carcinoma (NSCLC). The primary objective was survival. Secondary objectives were time to disease progression, response rates, evaluation of toxicity, disease-related symptoms, World Health Organization performance status (PS), and quality of life (QoL). METHODS. Three hundred seventy-two chemotherapy-naïve patients with International Staging System Stage III/IV NSCLC who were ineligible for curative radiotherapy or surgery were randomized to receive either 4 cycles of gemcitabine (1000 mg/m2 on Days 1, 8, and 15) plus carboplatin (area under the serum concentration-time curve, 5; given on Day 1) every 4 weeks (the GC arm) or MIC/MVP every 3 weeks (the MIC/MVP arm). RESULTS. There was no significant difference in median survival (248 days in the MIC/MVP arm vs. 236 days in the GC arm) or time to progression (225 days in the MIC/MVP arm vs. 218 days in the GC arm) between the 2 treatment arms. The 2-year survival rate was 11.8% in the MIC/MVP arm and 6.9% in the GC arm. The 1-year survival rate was 32.5% in the MIC/MVP arm and 33.2% in the GC arm. In the MIC/MVP arm, 33% of patients responded (4 complete responses [CRs] and 57 partial responses [PRs]) whereas in the GC arm, 30% of patients responded (3 CRs and 54 PRs). Nonhematologic toxicity was comparable for patients with Grade 3-4 symptoms, except there was more alopecia among patients in the MIC/MVP arm. GC appeared to produce more hematologic toxicity and necessitated more transfusions. There was no difference in performance status, disease-related symptoms, of QoL between patients in the two treatment arms. Fewer inpatient stays for complications were required with GC. CONCLUSIONS. The results of the current study failed to demonstrate any difference in efficacy between the newer regimen of GC and the older regimens of MIC and MVP. © 2003 American Cancer Society.

Fibrogenic stresses activate different mitogen-activated protein kinase pathways in renal epithelial, endothelial or fibroblast cell populations

Fibrogenic stresses promote progression of renal tubulointerstitial fibrosis, disparately affecting survival, proliferation and trans-differentiation of intrinsic renal cell populations through ill-defined biomolecular pathways. We investigated the effect of fibrogenic stresses on the activation of cell-specific mitogen-activated protein kinase (MAPK) in renal fibroblast, epithelial and endothelial cell populations. The relative outcomes (cell death, proliferation, trans-differentiation) associated with activation or inhibition of extracellular-regulated protein kinase (ERK) or stress activated/c-Jun N terminal kinase (JNK) were analysed in each renal cell population after challenge with oxidative stress (1 mmol/L H2O2), transforming growth factor-beta1 (TGF-beta1, 10 ng/mL) or tumour necrosis factor-alpha (TNF-alpha, 50 ng/mL) over 0-20 h. Apoptosis increased significantly in all cell types after oxidative stress (P < 0.05). In fibroblasts, oxidative stress caused the activation of ERK (pERK) but not JNK (pJNK). Inhibition of ERK by PD98059 supported its role in a fibroblast death pathway. In epithelial and endothelial cells, oxidative stress-induced apoptosis was preceded by early induction of pERK, but its inhibition did not support a pro-apoptotic role. Early ERK activity may be conducive to their survival or promote the trans-differentiation of epithelial cells. In epithelial and endothelial cells, oxidative stress induced pJNK acutely. Pretreatment with SP600125 (JNK inhibitor) verified its pro-apoptotic activity only in epithelial cells. Transforming growth factor-beta1 did not significantly alter mitosis or apoptosis in any of the cell types, nor did it alter MAPK activity. Tumor necrosis factor-alpha caused increased apoptosis with no associated change in MAPK activity. Our results demonstrate renal cell-specific differences in the activation of ERK and JNK following fibrotic insult, which may be useful for targeting excessive fibroblast proliferation in chronic fibrosis.

Weekly cisplatin concurrently with radiotherapy in head and neck squamous cell cancer : a retrospective analysis of a tertiary institute experience

Radiotherapy combined with three weekly 100 mg/m2 of cisplatin is the accepted standard of care in head and neck squamous cell carcinoma. However, this regimen is associated with severe toxicities with devastating effects on patients. Alternative protocols like weekly 40 mg/m2 have been used in an attempt to reduce toxicities. The main objective of the present study is to identify the dose intensities and toxicities of weekly cisplatin in patients treated in a tertiary centre over a 12 month period. Included patients had squamous cell carcinoma arising in the oral cavity, oropharynx, larynx, or hypopharynx. Patients were excluded if they had nasopharyngeal squamous cell carcinoma, distant metastasis or if they had prior treatment for head and neck cancer excluding neck dissection. During the study period, 52 patients met the inclusion criteria and their data were retrospectively obtained from the patients' database of St James hospital, Dublin. The median age of the study cohort was 54 years (range 33-73). Of the patients, 40 (76.9 %) were male and 12 (20.1 %) were female. The primary tumour sites were as follows: oral cavity and oropharynx in 38 (73 %), larynx in 10 (19 %), and hypopharynx in 4 (8 %). In total, 33 (63.5 %) patients had stage IV disease, while 19 (36.5 %) had stage III disease. Treatment was definitive in 35 (67 %) patients and adjuvant in 17 (35 %). Full-dose radiotherapy was achieved in 50 (96 %) patients. Only 22 (42.3 %) patients completed the intended six cycles of chemotherapy. Cumulative dose of 200 mg/m2 or more was reached in 37 (71 %) patients. The acute adverse effects included grades 3 and 4 mucositis, which occurred in 22 (43.3 %) and 6 patients (12 %), respectively. Grade 3 and 4 neutropenia occurred in six (11.5 %) and three (5.7 %) patients, respectively. The only other haematological toxicity was grade 3 anaemia in 20 (38.4 %) patients. There was no grade 3 or 4 renal toxicity among the study cohort, although grade 2 was observed in six (11.5 %) patients. Death occurred in one patient due to neutropenic septicaemia. In conclusion, weekly cisplatin is associated with moderate to severe toxicities and might lead to suboptimal chemotherapy delivery. More prospective clinical studies are required to determine the optimal chemoradiation regimen in head and neck squamous cell carcinoma.

Glutathione transferase activities in renal carcinomas and adjacent normal renal tissues : factors influencing renal carcinogenesis induced by xenobiotics

In general, the biological activation of nephrocarcinogenic chlorinated hydrocarbons proceeds via conjugatiton with glutathione. It has mostly been assamed that the main site of initial conjugation is the liver, followed by a mandatory transfer of intermediates to the kidney. It was therefore of interest to study the enzyme activities of subgroups of glutathione transferases (GSTs) in renal cancers and the surrounding normal renal tissues of the same individuals (n = 21). For genotyping the individuals with respect to known polymorphic GST isozymes the following substrates with differential specificity were used: 1-chloro-2,4-dinitrobenzene for overall GST activity (except GST θ); 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole for GST α; 1,2-dichloro-4-nitro-benzene for GST μ; ethacrynic acid and 4-vinylpyridine for GST π; and methyl chloride for GST θ. In general, the normal tissues were able to metabolize the test substrates. A general decrease in individual GST enzyme activities was apparent in the course of cancerization, and in some (exceptional) cases individual activities, expressed in the normal renal tissue, were lost in the tumour tissue. The GST enzyme activities in tumours were independent of tumour stage, or the age and gender of the patients. There was little influence of known polymorphisms of GSTM1, GSTM3 and GSTP1 upon the activities towards the test substrates, whereas the influence of GSTT1 polymorphism on the activity towads methyl chloride was straightforward. In general, the present findings support the concept that the initial GST-dependent bioactivation step of nephrocarcinogenic chlorinated hydrocarbons may take place in the kidney itself. This should be a consideration in toxicokinetic modelling.

Expression and activity profiles of DPP IV/CD26 and NEP/CD10 glycoproteins in the human renal cancer are tumor-type dependent

Background: Cell-surface glycoproteins play critical roles in cell-to-cell recognition, signal transduction and regulation, thus being crucial in cell proliferation and cancer etiogenesis and development. DPP IV and NEP are ubiquitous glycopeptidases closely linked to tumor pathogenesis and development, and they are used as markers in some cancers. In the present study, the activity and protein and mRNA expression of these glycoproteins were analysed in a subset of clear-cell (CCRCC) and chromophobe (ChRCC) renal cell carcinomas, and in renal oncocytomas (RO). Methods: Peptidase activities were measured by conventional enzymatic assays with fluorogen-derived substrates. Gene expression was quantitatively determined by qRT-PCR and membrane-bound protein expression and distribution analysis was performed by specific immunostaining. Results: The activity of both glycoproteins was sharply decreased in the three histological types of renal tumors. Protein and mRNA expression was strongly downregulated in tumors from distal nephron (ChRCC and RO). Moreover, soluble DPP IV activity positively correlated with the aggressiveness of CCRCCs (higher activities in high grade tumors). Conclusions: These results support the pivotal role for DPP IV and NEP in the malignant transformation pathways and point to these peptidases as potential diagnostic markers.

Cell cycle arrest and apoptosis in TP53 subtypes of bladder carcinoma cell lines treated with cisplatin and gemcitabine

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)