This Month in Investigative Urology

Article Outline

• Phosphatidylinositol-3-Kinase/Akt Signaling Pathway and Kidney Cancer
• Unilateral Ureteral Obstruction and Kidney Acid-Base Transporters
• Polycomb Group Protein Prognostic Relevance for RCC
• Murine Urothelial Carcinoma and Human Bacillus Calmette-Guerin Response
• Orthotopic Bladder Tumor Implantation in a Syngeneic Mouse Model
• Copyright

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Phosphatidylinositol-3-Kinase/Akt Signaling Pathway and Kidney Cancer 

Improved understanding of the biology of renal cell carcinoma (RCC) has permitted the development of novel targeted therapeutic agents that have changed the natural history of this disease. Nevertheless, advanced RCC remains an incurable disease and newer treatment options are urgently needed. Of the molecular pathways involved in RCC pathogenesis, the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway represents an appealing therapeutic target. This pathway is activated by many cellular stimuli, and it regulates fundamental cellular functions including transcription, translation, proliferation, growth and survival. It also closely interacts with many other key pathways such as mTOR and, thus, is linked to angiogenesis. Disturbed activation of the PI3K/Akt pathway is associated with many human malignancies.

Porta and Figlin (page 2569) from Duarte, California reviewed the available literature on PI3K/Akt and PI3K/Akt targeting drugs, searching MEDLINE® and the proceedings of the main oncological meetings to identify information concerning the role of this pathway in RCC pathogenesis as well as data on the preclinical and clinical activity of compounds specifically targeting this pathway. They found evidence linking PI3K/Akt alterations with RCC. Thus, RCC is an ideal setting in which to test compounds specifically targeting this pathway. Several PI3K/Akt inhibitors are currently in preclinical and early clinical development but only perifosine appears to be at a more advanced stage, having been tested with promising results alone or combined with other molecularly targeted agents. The authors conclude that the PI3K/Akt pathway has a pivotal role in RCC pathogenesis and, thus, represents an ideal target for therapeutic intervention. Perifosine has proved to be clinically active and should be considered an extremely interesting drug to be used alone or in combination.

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Unilateral Ureteral Obstruction and Kidney Acid-Base Transporters 

Unilateral ureteral obstruction (UUO) is a common clinical problem that is often associated with a form of renal tubular acidosis characterized by the inability to lower urinary pH maximally (less than 5.5) under the stimulus of systemic acidemia. The defect is caused by decreased net H⁺ secretion and/or HCO₃⁻ reabsorption. Wang et al (page 2964) from Aarhus, Denmark studied the changes in expression of key renal acid-base transporters in obstructed and nonobstructed kidneys in response to UUO and release of obstruction to identify the underlying cellular and molecular mechanisms for the acidification defect associated with UUO.

Expression levels of key acid-base transporters were examined along the renal nephron segments and collecting duct in Wistar rats subjected to 24-hour UUO, release of UUO followed for 4 days (UUO-R) or UUO-R plus experimental acidosis induced by oral administration of NH₄Cl (UUO-A). Using semiquantitative immunoblotting they found that UUO caused significant mean ± SE down-regulation of type 3 Na⁺/H⁺ exchanger (NHE3) to 53% ± 9%, electrogenic Na⁺/HCO₃⁻ cotransporter (NBC1) to 60% ± 9%, type 1 bumetanide sensitive Na⁺-K⁺(NH₄⁺)-2Cl⁻ cotransporter (NKCC2) to 64% ± 7%, electroneutral Na⁺/HCO₃⁻ cotransporter (NBCn1) to 43% ± 4% and anion exchanger (pendrin) to 53% ± 10% in the obstructed kidney. These findings were confirmed by immunohistochemistry. After release of UUO, down-regulation of these transporters persisted with marked down-regulation of H⁺-adenosine triphosphatase in the obstructed kidney. In UUO-A rats plasma pH and HCO₃⁻ levels were dramatically reduced in response to NH₄Cl for 2 days compared with those in sham operated rats, indicating a defect in H⁺ excretion and HCO₃⁻ reabsorption after the release of obstruction. Expression of these transporters did not change in the contralateral nonobstructed kidney of UUO and UUO-R rats. The authors conclude that the expression of renal acid-base transporters is markedly reduced in the obstructed kidney after UUO, which may be responsible for impaired renal H⁺ excretion and HCO₃⁻ reabsorption contributing to the urinary acidification defect in response to UUO.

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Polycomb Group Protein Prognostic Relevance for RCC 

Renal cell carcinoma is the third most common urogenital malignancy. It is highly resistant to conventional chemotherapy and radiotherapy, resulting in a poor outcome for patients with advanced or metastatic disease. The investigation of molecular factors that may have a role in RCC tumor biology has the potential not only to identify markers that better reflect individual tumor behavior, but also to provide targets for novel therapeutic approaches.

The human homologue of the Drosophila protein Enhancer of Zeste 2 (EZH2) increases the proliferation rate and apoptosis resistance of RCC cell lines. Hinz et al (page 2920) from Berlin, Germany studied the association of EZH2 expression with histopathological features and disease outcomes in a large cohort of patients who underwent surgery for RCC. They used real-time reverse transcriptase-polymerase chain reaction to quantify EZH2 expression in malignant and adjacent benign renal tissue from a cohort of 119 patients with clear cell RCC. Immunohistochemistry was performed in a subset of samples. The impact of EZH2 expression on clinicopathological tumor features and outcome was investigated. EZH2 was over expressed in RCC (median 57.02, range 0 to 368.11) compared to adjacent benign renal parenchyma (median 0, range 0 to 280.87) (p <0.001). Immunohistochemistry showed concordant results and revealed EZH2 protein predominantly located in the nucleus. EZH2 expression was not associated with histopathological tumor features and patient characteristics. High EZH2 levels were predictive of a lower disease recurrence rate on univariate and multivariate analysis (p=0.047 and p=0.037, respectively). The authors suggest their data support a role of EZH2 expression for RCC tumorigenesis rather than tumor progression, and that high EZH2 tumor levels appear to indicate less aggressive tumor phenotypes with a favorable prognosis in RCC. EZH2 may not only provide a potential therapeutic target, but also a molecular parameter for outcome prediction in patients with RCC.

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Murine Urothelial Carcinoma and Human Bacillus Calmette-Guerin Response 

Bacillus Calmette-Guerin (BCG) exposure alters gene expression and the biological phenotype of human urothelial carcinoma (UC) cells. For example, BCG activates multiple intracellular signaling pathways that regulate downstream gene expression. While the contribution of these effects to the antitumor activity of BCG is unclear, the nature of BCG induced changes supports a potential role. In contrast to in vitro studies, in vivo animal model systems have the potential to provide more robust, clinically relevant insight into the BCG mechanism of action. The mouse UC cell line MB49 is widely used as an in vitro and in vivo model of UC. Little comparative data exist on the molecular and phenotypic responses of this cell line relative to human cell lines.

Chen et al (page 2932) from Milwaukee, Wisconsin compared the effect of BCG on the MB49 cell line relative to responses previously observed in the human UC lines T24 and 253J. The molecular end points in MB49 cells after BCG exposure included signaling pathway activation (nuclear factor κB [NFκB], AP1, C/EBP), gene expression (interleukin-6 and p21), high molecular group box protein 1 (HMGB1) release/responsiveness and gene expression profiling at 6 hours. Phenotypic response end points were direct cytotoxicity using dye exclusion, viability on MTT assay, apoptotic sensitivity and cell cycle compartmentalization. NFκB, AP1, C/EBP, interleukin-6 and p21 reporter constructs were activated in MB49 cells in response to BCG. Gene expression profiles demonstrated an inflammatory/immune clustering response. BCG treatment decreased cell viability and induced G1 cell cycle arrest. Treatment of MB49 cells with BCG induced caspase independent cell death while simultaneously decreasing sensitivity to proapoptotic agents. Cell death was associated with release of the necrotic cell death marker HMGB1. MB49 cells expressed HMGB1 receptors and activated intracellular NF-κB signaling pathways in response to BCG. The authors conclude that MB49 cells demonstrate molecular and phenotypic responses to BCG that replicate those observed in human UC lines, and that the MB49 cell line is an excellent model in which to assess the complex in vivo interrelationships among tumor, host and BCG.

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Orthotopic Bladder Tumor Implantation in a Syngeneic Mouse Model 

Bladder cancer is the fourth most common malignancy in men and the ninth most common in women in the United States. In general, animal models such as those of orthotopic bladder cancer are important in investigating the pathogenesis of cancer and evaluating the effects of different therapeutic interventions. Chan et al (page 2926) from Cleveland, Ohio established a reliable technique for the orthotopic implantation of bladder tumor cells in a syngeneic mouse model. They implanted MBT-2 murine bladder cancer cells transurethrally into the bladder of syngeneic C3H/HeJ mice. Different chemical pretreatment methods were used before tumor implantation including phosphate buffered saline (control), hydrochloric acid, trypsin and poly-L-lysine (PLL). Bladder tumor cells were instilled into the intravesical space after chemical pretreatment. Tumor take and bladder tumor volume were determined by micro ultrasound imaging. Bladders were harvested at the end of the study for measurement of bladder weight and for histopathological examination. The authors discontinued bladder pretreatment with hydrochloric acid (in 5) due to significant adverse reactions resulting in 1 mouse death, and 2 animals with severe bladder inflammation and hematuria 3 days after pretreatment. Pretreatment with phosphate buffered saline, trypsin and PLL in 6 animals each was tolerated well without significant adverse reactions or mortality. The tumor take rate in the control, trypsin and PLL pretreatment groups was 33%, 83% and 83%, respectively. The tumor take rate was higher in mice instilled with 2 × 10⁶ cells than in those with 1 × 10⁶ cells (93% vs 73%, p <0.05). The authors suggest that their method is reliable and feasible, and that it is possible to orthotopically implant bladder tumor cells into a syngeneic mouse model. The model may be suitable for the evaluation of treatment paradigms for bladder cancer.