N-myc downstream-regulated gene 1 can promote vasculogenic mimicry and angiogenesis in urothelial carcinoma.

Urothelial carcinoma (UC) of the bladder is a common cause of cancer-related death worldwide. Vasculogenic mimicry (VM) is a process by which the malignant cells can generate vascular-like structures formed of periodic acid-Schiff (PAS) positive/CD31 negative extracellular matrix independent of angiogenesis and thus promotes tumor progression. N-myc downstream-regulated gene 1 (NDRG1) is a protein that can modulate tumor angiogenesis; however, its role in regulating tumor angiogenesis and VM formation has not been previously investigated in UC. This study aims to evaluate the role of intra-tumor microvessel density (MVD) (as a surrogate measure of angiogenesis), VM, and NDRG1 in UC and their correlation with different clinicopathologic features, then assess the correlation between them in UC. Sixty specimens of UC of the bladder were included. PAS-CD31 immunohistochemical double staining method was used to evaluate the intra-tumor MVD and VM. Immunohistochemical expression of NDRG1 was also examined. VM and NDRG1 expression were detected in 41.7% and 83.3% of UC specimens respectively. The mean of intra-tumor MVD, VM area, and NDRG1 was significantly higher in tumors with higher grade, lymphovascular invasion, and higher T stage. NDRG1 expression was positively correlated with MVD and VM. We can suggest that MVD, VM, and NDRG1 may serve as poor prognostic markers for UC. The positive correlation between NDRG1 and both MVD and VM may provide the first evidence that NDRG1 can induce tumor angiogenesis and VM in UC which may offer a novel pathway for further therapeutic strategies.

PKM2 Is Essential for Bladder Cancer Growth and Maintenance.

Pyruvate kinase M2 (PKM2) has been shown to promote tumorigenesis by facilitating the Warburg effect and enhancing the activities of oncoproteins. However, this paradigm has recently been challenged by studies in which the absence of PKM2 failed to inhibit and instead accelerated tumorigenesis in mouse models. These results seem inconsistent with the fact that most human tumors overexpress PKM2. To further elucidate the role of PKM2 in tumorigenesis, we investigated the effect of PKM2 knockout in oncogenic HRAS-driven urothelial carcinoma. While PKM2 ablation in mouse urothelial cells did not affect tumor initiation, it impaired the growth and maintenance of HRAS-driven tumors. Chemical inhibition of PKM2 recapitulated these effects. Both conditions substantially reduced complex formation of PKM2 with STAT3, their nuclear translocation, and HIF1α- and VEGF-related angiogenesis. The reduction in nuclear STAT3 in the absence of PKM2 also correlated with decreased autophagy and increased apoptosis. Time-controlled, inducible PKM2 overexpression in simple urothelial hyperplasia did not trigger tumorigenesis, while overexpression of PKM2, but not PKM1, in nodular urothelial hyperplasia with angiogenesis strongly accelerated tumorigenesis. Finally, in human patients, PKM2 was overexpressed in low-grade nonmuscle-invasive and high-grade muscle-invasive bladder cancer. Based on these data, PKM2 is not required for tumor initiation but is essential for tumor growth and maintenance by enhancing angiogenesis and metabolic addiction. The PKM2-STAT3-HIF1α/VEGF signaling axis may play a critical role in bladder cancer and may serve as an actionable therapeutic target. SIGNIFICANCE: Genetic manipulation and pharmacologic inhibition of PKM2 in mouse urothelial lesions highlight its essential role in promoting angiogenesis and metabolic addiction, events indispensable for tumor growth and maintenance.

Perifocal edema volume is not associated with immunohistochemical features reflecting proliferation potential, microvessel density, neoangiogenesis and invasiveness in brain metastasis.

OBJECTIVE: Perifocal edema of brain tumors is associated with survival and neurological symptoms. Our aim was to analyze associations between perifocal edema and immunohistochemical features including proliferation potential, microvessel density, neoangiogenesis and invasiveness in brain metastasis (BM). METHODS: 35 patients with BM were included into the retrospective study. The tumors were localized supratentorial in 25 lesions (71.4%) and infratentorial in 10 lesions (28.6%). The following immunohistochemical features were calculated on histopathological specimens: microvessel density, proliferation index Ki 67, matrix-metallopeptidase 9 (MMP9) extracellular matrix metalloproteinase inducer (EMMPRIN) and vascular endothelial growth factor (VEGF) expression. Tumor and edema volumes were estimated semiautomatically on magnetic resonance images. RESULTS: There were no correlations between tumor volume and edema volume. Moreover, no correlation was identified between the investigated immunohistochemical features and tumor/edema volume. In the non-small cell lung cancer subgroup, a positive correlation between tumor volume and VEGF expression was observed (r = 0.52, P = 0.02) and edema volume correlated inversely with MMP9 expression (r = -0.53, P = 0.02). CONCLUSION: In BM, no linear associations exist between tumor volumes, edema volumes and immunohistochemical features reflecting proliferation potential, neoangiogenesis, microvessel density and MMP9 expression. However, in the subgroup of non-small cell lung cancer, there might be associations between MMP9 expression and edema volume as well as between tumor volume and angiogenesis.

Contrast-Enhanced Ultrasound Characteristics of Renal Pelvis Urothelial Carcinoma and Its Relationship with Microvessel Density.

We studied the characteristics of contrast-enhanced ultrasound (CEUS) in renal pelvic urothelial carcinomas and explored its performance in assessing microvessel density (MVD) of tumor tissues. We retrospectively analyzed the characteristics of 125 cases, which were confirmed pathologically to be renal pelvic urothelial carcinomas using CEUS. We performed CEUS and found that most tumors presented with an enhanced mode of "slow-in (mean = 16.7 ± 2.6 s, range: 12-25 s), hypo-enhancement and fast-out (mean = 69.3 ± 16.2 s, range: 42-113 s)." However, the wash-in pattern, homogeneity and wash-out pattern observed with CEUS was not correlated with pT stage and grade (p > 0.05). But advanced-pT-stage and high-grade tumors had a higher peak enhancement than early-pT-stage and low-grade tumors (p < 0.01). Peak enhancement obtained with CEUS can be used to evaluate the pT stage and grade of renal pelvic urothelial carcinomas more effectively. The MVD of those tissues was observed using immunohistochemical staining of cluster of differentiation 34 (CD34). MVD in the advanced-pT-stage and high-grade groups was significantly higher than that in the early-pT-stage and low-grade groups (p < 0.01). As tumor pT stage and grade improved, CEUS peak enhancement intensity and MVD of tumors also exhibited an upward trend. CEUS peak enhancement intensity has the potential to determine MVD of renal pelvic urothelial carcinomas.

Volumetric imaging: a potential tool to stage upper tract urothelial carcinoma.

PURPOSE: To investigate whether volumetric imaging of tumor vasculature can be used to phenotypically characterize advanced upper tract urothelial carcinoma, and if this technique can distinguish aggressive invasive tumors from non-aggressive superficial ones. METHODS: In a pilot study, two TaG1 and two T3G3 formalin-fixed paraffin-embedded (FFPE) tumor samples were examined using the DIPCO pipeline (Tanaka et al. in Nature Biomed Eng 1(10):796-806. https://doi.org/10.1038/s41551-017-0139-0 , 2017). Briefly, punch biopsies of FFPE tumors were deparaffinized, cleared, immunolabeled with the vessel marker CD34 and imaged with a light-sheet microscope. Thereafter, the three-dimensional (3D) vasculature of the tumors was analyzed and characterized using a specialized image processing software. RESULTS: We found that T3G3 tumors had increased CD34 density kurtosis and skewness compared to TaG1 tumors. This suggests that analysis of the 3D vasculature can distinguish between high-grade invasive and low-grade superficial tumors. CONCLUSIONS: Volumetric imaging of tumor samples may represent novel methodology that can complement conventional histopathology. Volumetric imaging enabled us to differentiate between invasive and non-invasive upper tract urothelial carcinoma. The method is of particular interest in diagnostic work-up of patients with upper tract urothelial carcinoma as previous findings indicate that volumetric imaging of vascular patterns could be used to differentiate superficial and invasive urothelial carcinoma, irrespective of if the tumor sample was deep or superficial. However, further and more extensive studies are required before this method can be applied clinically.

Effectiveness of the combination of vascular targeted photodynamic therapy and anti-cytotoxic T-lymphocyte-associated antigen 4 in a preclinical mouse model of urothelial carcinoma.

OBJECTIVE: To investigate the effectiveness of combination treatment of vascular targeted photodynamic therapy and anti-cytotoxic T-lymphocyte-associated antigen 4 immunotherapy in a mouse model of urothelial carcinoma. METHODS: We used C57BL/6 mice injected with murine bladder 49 cell line. Mice were randomly allocated into four treatment groups: vascular targeted photodynamic therapy only, anti-cytotoxic T-lymphocyte-associated antigen 4 only, combination therapy and control. We carried out three separate experiments that used distinct cohorts of mice: tumor growth and development of lung metastases monitored with bioluminescent imaging (n = 91); survival evaluated with Kaplan-Meier curves (n = 111); and tumor cell population studied with flow cytometry (n = 20). In a fourth experiment, we re-challenged tumors in previously treated mice and compared tumor growth with that of naïve mice. RESULTS: Combination therapy provided significant benefits over the other three treatment groups: prolonged survival (P < 0.0001), lower tumor signal (P < 0.0001) and decreased lung signal uptake (P ≤ 0.002). We also observed that mice previously treated with vascular targeted photodynamic therapy only or combination therapy did not present tumor growth after re-challenged tumors. CONCLUSIONS: Combination of vascular targeted photodynamic therapy with anti-cytotoxic T-lymphocyte-associated antigen 4 is an effective therapy in a urothelial carcinoma syngeneic mouse model. The present results suggest this therapy as a potential treatment option for both bladder and upper tract tumors in future clinical trials.

Novel Molecular Targets for the Therapy of Urothelial Cancer.

First-line platinum-based chemotherapy combinations are considered standard-of-care in locally advanced and metastatic urothelial cancer. However, long-term outcomes, including disease-specific and overall survival, remain poor. In addition, a number of patients with advanced urothelial carcinoma have co-existing medical issues that preclude the use of conventional chemotherapy. Improvements in our understanding over the molecular mechanisms of urothelial cancer have led to first-generation clinical trials evaluating novel agents targeting molecular pathways that may be relevant, at least in sub-populations. Emerging information regarding outcome with agents targeting novel molecular targets in advanced urothelial cancer is discussed in this review.

Expression of pigment epithelium-derived factor and tumor necrosis factor-α is correlated in bladder tumor and is related to tumor angiogenesis.

OBJECTIVE: Angiogenesis is a pivotal process on which solid tumor growth is substantially dependent. Pigment epithelium-derived factor (PEDF) is the most potent natural anti-angiogenic factor, which has seldom been studied in bladder tumor, and whose functioning pathway remains unclear. We have thus investigated PEDF expression in relation to tumor necrosis factor-α (TNF-α) and microvessel density (MVD) with immunohistochemistry. METHODS: Antibodies of PEDF and TNF-α were examined by Western blotting before immunohistochemistry. Sixty-four urothelial tumor sections and 23 normal controls were stained and expression of PEDF, TNF-α, and MVD were studied. RESULTS: Decreased PEDF expression and increased TNF-α expression was noticed in tumorous tissue compared with healthy urothelium. Lower PEDF expression was related to higher tumor grade but stage. Increased TNF-α expression was noticed in recurrent, larger tumors as well as in tumors with progression in grade and stage. Expression of PEDF and TNF-α was correlated in bladder tumor. PEDF or TNF-α was correlated with MVD negatively or positively, respectively, in cancerous tissue and tumorous grouping without correlation in papillary urothelial neoplasm of low malignant potential. CONCLUSION: Expressional change of PEDF and TNF-α is in relation to angiogenesis of bladder tumor, especially in bladder cancer development.

Angiogenesis in upper tract urothelial carcinoma associated with Balkan endemic nephropathy.

Upper tract urothelial carcinoma (UTUC) associated with Balkan endemic nephropathy (BEN) is characterized by a number of aberrations in cell-cycle regulation and apoptosis. The aim of this study was to detect angiogenesis-related marker(s) specific for BEN UTUC, and to examine the influence of HIF 1α upon angiogenesis and apoptosis in UTUC. Present investigation included 110 patients with UTUC, 50 from BEN region and 60 control tumors. Altered expression of VEGFR1 was more often present in control UTUC than in BEN tumors (p<0.005). It was associated with high grade, low and high stage, solid growth, and metaplastic change of control UTUC. Microvessel density assessed by CD31 (MVD CD31) was significantly higher in UTUC with lymphovascular invasion (p<0.05), and in BEN tumors with papillary growth (p<0.05). Discriminant analysis indicated that BEN and control tumors do not differ significantly in expression of angiogenesis related markers. The most important discriminant variable that determined control UTUC was expression of VEGFR1 (p=0.002). HIF 1α in UTUC significantly correlated with the low stage, papillary growth and expression of Bcl-2, Caspase-3 index, and MVD CD34 (p<0.001; 0.0005; 0.01; 0.005; 0.01, respectively). HIF-1α may be helpful marker in evaluation of UTUC, especially when combined with angiogenesis and apoptosis.

Expression of miRNAs involved in angiogenesis, tumor cell proliferation, tumor suppressor inhibition, epithelial-mesenchymal transition and activation of metastasis in bladder cancer.

PURPOSE: miRNAs are noncoding RNAs that posttranscriptionally regulate gene expression. Altered expression and function have been observed in bladder cancer. We analyzed the expression profile of a group of miRNAs involved in bladder cancer angiogenesis, tumor cell proliferation, tumor suppressor inhibition, epithelial-mesenchymal transition and metastasis activation. Prognostic and diagnostic value, and validated targets were further examined. MATERIALS AND METHODS: Using quantitative real-time polymerase chain reaction 77 bladder cancer cases and 77 matched tumor associated normal samples were investigated to determine the expression of miR-10b, 19a, 19b, 21, 126, 145, 205, 210, 221, 296-5p and 378. The relationship between miRNA expression, patient survival and tumor pathological features was also examined. RESULTS: miR-10b, 19a, 126, 145, 221, 296-5p and 378 were significantly down-regulated in bladder cancer compared to adjacent normal urothelium. miR-145 was the most down-regulated microRNA of this group. miR-19b, 21, 205 and 210 showed no significant difference between the 2 tissue types. High miR-21 expression correlated with worse overall patient survival (p = 0.0099). Multivariate analysis revealed that miR-21, 210 and 378 may serve as independent prognostic factors for overall patient survival (p = 0.005, 0.033 and 0.012, respectively). miR-21 and 378 may serve as independent prognostic factors for recurrence (p = 0.030 and 0.031, respectively). miR-145, 221, 296-5p and 378 showed the best combined ROC curves for specificity and sensitivity. miRWalk analysis was used to identify validated miRNA target genes. Further Gene Ontology enrichment revealed the main classes of biological functions of these validated targets. CONCLUSIONS: Most miRNAs analyzed are down-regulated in bladder cancer. They may serve as candidate biomarkers for diagnostic and prognostic purposes in the future.

[Therapy for non-muscle invasive bladder cancer: HP-NAP]. / La risposta immunitaria Th1 mediata nella terapia della neoplasia vescicale non muscolo invasiva: il ruolo di HP-NAP.

PURPOSE: Patients with non-muscle invasive bladder cancer recurrence after 2 induction courses of BCG are eligible for radical cystectomy. So, in the last years research to discover new drugs for the management of non-muscle invasive bladder cancer recurrence after failure of first and second line therapy is ongoing. In accordance to the results obtained with BCG, whose mechanism depends on the induction of the T helper 1 (TH1) immune response, we investigated the activity of a Toll-like receptor (TLR) 2 ligand, named Helicobacter Pylori Neutrophil Activating Protein (HP-NAP), that we recently demonstrated being able of enhancing the differentiation of Th1 cells, both in vitro and in vivo, because of its ability to create an IL-12 enriched milieu. MATERIALS AND METHODS: We show here, in a mouse model of bladder neoplasm implants, that local administration of HP-NAP decreases tumor growth by inducing tumor necrosis. RESULTS: The result is joined up with a massive cluster of both CD4+ and CD8+ IFN-γ+ cells, within neoplasm and regional lymph nodes. It is of note that HP-NAP-treated tumors show also a reduced vascularization due to the anti-angiogenic activity of IFN-γ induced by HP-NAP. CONCLUSIONS: The present study suggests that the activity of HP-NAP against urothelial tumor burden warrants subsequent in vivo studies.

Phase II study of sunitinib as first-line treatment of urothelial cancer patients ineligible to receive cisplatin-based chemotherapy: baseline interleukin-8 and tumor contrast enhancement as potential predictive factors of activity.

BACKGROUND: A strong rationale supports the role of antiangiogenic drugs in urothelial cancer. This trial was designed to assess the activity of sunitinib as first-line treatment in patients with metastatic urothelial cancer ineligible for cisplatin and to explore molecular and imaging variables predictive of clinical benefit. PATIENTS AND METHODS: This was a multicenter phase II trial with sunitinib 50 mg daily in 4/2-week schedule. Eligibility criteria were as follows: creatinine clearance 30-60 ml/min, Eastern Cooperative Oncology Group Pperformance Sstatus of one or less, and adequate hepatic and hematologic function. Twelve circulating cytokines were evaluated at baseline and sequentially using Luminex xMAP(®) (Austin, TX). Baseline and treatment-related changes in perfusion were evaluated in a patient subgroup using contrast-enhanced computed tomography. RESULTS: On intention-to-treat analysis, 38 patients showed 3 (8%) partial responses (PRs) and 19 (50%) presented with stable disease (SD), 17 (45%) of them ≥3 months. Clinical benefit (PR + SD) was 58%. Median time to progression (TTP) was 4.8 months and median overall survival 8.1 months. Toxicity was consistent with previous reports for sunitinib. Low interleukin-8 (IL-8) baseline levels were significantly associated with increased TTP. Baseline tumor contrast enhancement with >40 Hounsfield units was associated with clinical benefit. CONCLUSIONS: This study highlights the potential role of the angiogenic pathway as a therapy target in urothelial cancer. Baseline IL-8 serum levels and contrast enhancement of lesions warrant further study.

[Evaluation of HER2 protein overexpression in non-muscle-invasive bladder cancer with emphasis on tumour grade and recurrence]. / Evaluación de la sobreexpresión de proteína HER2 en cáncer vesical no músculo invasivo con énfasis en el grado tumoral y en la recurrencia.

OBJECTIVE: To evaluate the prognostic value of HER2 expression in non-muscle invasive bladder transitional cell carcinoma (TCC) with special emphasis in the high grade population. MATERIALS AND METHODS (PATIENTS): Tissue microarrays (TMA) were performed with representative TUR-B specimens from 84 patients with non-muscle invasive bladder TCC (40 pT1GII and 44 pT1GIII) treated in our institution. Depth of invasion and grade were uniformly assigned by the same pathologist who performed blind immunohistochemical analysis with Hercep test: 3+ was considered strong positive HER2 overexpression. Other clinico-pathological variables were also assessed. RESULTS: HER2 protein overexpression was detected in 30/44 (68.2%) pT1GIII lesions and predicted recurrence in this subgroup of bladder TCC (p<0.01). Negative HER2 expression was detected in 26/40 (65%) cases with pT1GII TCC, and this condition was more frequent in unifocal tumours, without angiogenesis, with low recurrence rate and without progression. Recurrence-free survival can also be anticipated by HER 2 expression within pT1GII tumours (p<0.01). CONCLUSION: HER2 expression using Hercep test may be useful to predict recurrence in non-muscle invasive bladder TCC. The potential application of this study, especially regarding prediction of response to BCG, should be prospectively confirmed in multi-institutional trials.

Value of preoperative superselective embolization of the isthmus in a patient with upper urinary tract urothelial carcinoma and horseshoe kidney.

Standard treatment for upper urinary tract urothelial carcinoma (UUTUC) implies the radical removal of all urothelium-lined tissue, which requires nephroureterectomy with bladder cuff removal. We report on a patient with a rare coincidence of UUTUC and horseshoe kidney in whom a preoperative angiography helped to identify and subsequently embolize an abberant isthmic feeding artery, which was located in between both collecting systems. Ischemic discoloration of the isthmus area facilitated resection and no major blood loss occurred. Preoperative superselective embolization of the isthmus as the renal split area can be an effective tool to facilitate nephroureterectomy in the case of a horseshoe kidney.

Cyclooxygenase-2 and survivin in superficial urothelial carcinoma of the bladder and correlation with intratumoural microvessel density.

This study was designed to investigate the protein levels of cyclooxyogenase-2 (COX-2) and survivin in superficial urothelial carcinoma (UC) and their correlation with microvessel density (MVD). High-grade UC was positive for both COX-2 and survivin protein, and the proportion of tumours positive for both proteins increased with increasing tumour grade. The presence of COX-2 protein was significantly correlated with the presence of survivin protein. Both COX-2 and survivin positivity were significantly correlated with MVD in all patients regardless of tumour grade, but there was no correlation between MVD and COX-2 and survivin positivity by individual tumour grade. Although there was no significant difference in the proportion of COX-2-positive tumours when patients were stratified by tumour stage, a significantly higher proportion of patients with pT1 stage tumours were survivin-positive compared with patients with pTa stage tumours. COX-2 and survivin positivity were significantly correlated in all patients regardless of tumour grade or stage. COX-2 and survivin were significantly correlated in patients with pTa, but there was no correlation in pT1 tumours. These findings demonstrate that together, COX-2, survivin and MVD may play an important role in UC.

Large-scale real-time reverse transcription-PCR approach of angiogenic pathways in human transitional cell carcinoma of the bladder: identification of VEGFA as a major independent prognostic marker.

BACKGROUND: Actors of the angiogenesis pathways are targets for the new promising targeted therapies already used in several malignancies. In bladder cancer, antiangiogenic molecules could also add to already existing treatment options. OBJECTIVE: To evaluate the involvement of angiogenesis pathways in bladder carcinogenesis and identify new molecular markers having a clinical implication. DESIGN, SETTING, AND PARTICIPANTS: Expression levels of 40 genes involved in angiogenesis were assessed by quantitative real time RT-PCR in 157 urothelial tumour bladder samples obtained from patients who underwent transurethral bladder resection or radical cystectomy between 2001 and 2005. Pathologic tumour staging showed: 73 non-muscle-invasive bladder tumours (30 low-grade pTa, 14 high-grade pTa, and 29 high-grade pT1), and 84 muscle-invasive tumours (> or = pT2), all of high grade. RT-PCR results were associated with a survival analysis. RESULTS AND LIMITATIONS: VEGFA, MET, CXCR4, and IL8 were significantly overexpressed in tumour samples as compared to normal bladder tissue. VEGFA overexpressions were found in 89% of non-muscle-invasive and 66% of muscle-invasive tumour samples. In univariate analysis, for invasive tumours, VEGFA overexpression was associated with a poorer outcome in both overall and disease-free survival (p=0.011 and 0.026 respectively) at a 13-mo median follow-up. Multivariate analysis retained T stage, N status, and VEGFA overexpression as independent prognostic factors in both overall and disease-free survival (p=0.02 and p=0.04, respectively, for VEGFA). CONCLUSIONS: This study shows that, in bladder cancer, VEGFA status could be used as a prognostic factor at the individual level. VEGFA overexpression could guide a rationalized use of the costly antiangiogenic therapies which could therefore become part of the treatment options in bladder cancer.

Microvessel density (MVD) and cyclooxygenase-2 (COX-2)/ beta-catenin interaction are associated with relapse in patients with transitional carcinoma receiving adjuvant chemotherapy with paclitaxel/carboplatin: a hellenic cooperative oncology group (HECOG) study.

BACKGROUND: Cycloxygenase (COX)-2 has been associated with proliferation, apoptosis and angiogenesis in urothelial cancer. The prognostic significance of COX-2 in patients who received adjuvant chemotherapy for urothelial cancer was examined. PATIENTS AND METHODS: Expression of COX-2, p53, ki67, beta-catenin, vascular endothelial growth factor (VEGF) and microvessel density (MVD) were studied retrospectively in 59 patients with urothelial cancer (pT3, pT4, N+) who had undergone surgery. The patients had subsequently received adjuvant chemotherapy. RESULTS: Thirty-eight out of 59 cases (64%) were positive for COX-2. COX-2 was not associated either with progression-free survival (PFS) or overall survival (OS). MVD levels > or =47 were associated with longer median PFS compared with lower levels (not reached vs. 13 months [95% CI: 8-18], p=0.048). The median PFS for patients with beta-catenin nuclear accumulation and COX-2 expression was 6 months (95% CI: 4-7) compared with 19 months (95% CI: 14-23) for neither or only one of these factors (p=0.018). CONCLUSION: MVD may be a useful indicator of relapse in high-risk urothelial cancer treated with adjuvant chemotherapy.

Mitochondrial cytochrome B gene mutation promotes tumor growth in bladder cancer.

Mitochondria-encoded Cytochrome B (CYTB) gene mutations were reported in different cancers, but the effect of these mutations on cellular metabolism and growth is unknown. In a murine xenograft and human model of bladder cancer, we show the functional effect of overexpression of a 21-bp deletion mutation (mt) of CYTB. Overexpression of mtCYTB generated increased reactive oxygen species (ROS) accompanied by increased oxygen consumption and lactate production. MtCYTB overexpression induced significant tumor growth in vitro and in vivo by triggering rapid cell cycle progression through up-regulation of the nuclear factor-kappa B2 signaling pathway. Tumor-generated ROS induced in vitro lysis of normal splenocytes. Thus, we present physiologic and functional evidence for the role of a bona fide mitochondrial gene mutation in cancer.

Hypoxia-inducible factor-1alpha expression correlates with focal macrophage infiltration, angiogenesis and unfavourable prognosis in urothelial carcinoma.

BACKGROUND: Hypoxia inducible factor (HIF)-1alpha is a critical regulatory protein of cellular response to hypoxia and is closely related to angiogenic process. AIMS: To explore the potential role and the prognostic value of HIF-1alpha in urothelial carcinoma (UC). METHODS: Clinicopathological and follow-up data on 99 UC cases were reviewed and immunostained for HIF-1alpha, CD68, vascular endothelial growth factor (VEGF) and CD34 antigen. Tumour-associated macrophage (TAM) counts and HIF-1alpha expression were compared with clinicopathologic characteristics, overall survival (OS) and disease-free survival rates (DFS). RESULTS: High expression of HIF-1alpha was detected in 55 of 99 (55.6%) tumours. HIF-1alpha expression was correlated with tumour size, histological grade, tumour invasiveness and recurrence. VEGF and microvessel density (MVD) demonstrated their positive correlation with HIF-1alpha overexpression, supporting the correlation of HIF-1alpha up-regulation with tumour angiogenesis. Higher TAM infiltration was identified in high expression of HIF-1alpha cases rather than HIF-1alpha low expression cases (p = 0.002). Kaplan-Meier analysis found that HIF-1alpha overexpression and high TAM count was only associated with worse DFS (p = 0.009, p = 0.023) but was not associated with OS (p = 0.696, p = 0.141). Multivariate analyses indicated only tumour size (p = 0.038) to be an independently significant prognostic factor for OS, in addition, HIF-1alpha expression (p = 0.011), as well as histological grade (p = 0.038), and MVD (p = 0.004), to be independently significant prognostic factors for DFS. CONCLUSIONS: Our results indicate that HIF-1alpha is a key regulator of the angiogenic cascade. We show that HIF-1alpha is an independent prognostic factor for disease-free survival.