Gennady Bratslavsky

Asymmetric Hsp90 N Domain SUMOylation Recruits Aha1 and ATP-Competitive Inhibitors

The stability and activity of numerous signaling proteins in both normal and cancer cells depends on the dimeric molecular chaperone heat shock protein 90 (Hsp90). Hsp90s function is coupled to ATP binding and hydrolysis and requires a series of conformational changes that are regulated by cochaperones and numerous posttranslational modifications (PTMs). SUMOylation is one of the least-understood Hsp90 PTMs. Here, we show that asymmetric SUMOylation of a conserved lysine residue in the N domain of both yeast (K178) and human (K191) Hsp90 facilitates both recruitment of the adenosine triphosphatase (ATPase)-activating cochaperone Aha1 and, unexpectedly, the binding of Hsp90 inhibitors, suggesting that these drugs associate preferentially with Hsp90 proteins that are actively engaged in the chaperone cycle. Importantly, cellular transformation is accompanied by elevated steady-state N domain SUMOylation, and increased Hsp90 SUMOylation sensitizes yeast and mammalian cells to Hsp90 inhibitors, providing a mechanism to explain the sensitivity of cancer cells to these drugs.

Renal Mass Biopsy: Always, Sometimes, or Never?

Renal mass biopsy is a useful clinical tool; nevertheless, in a majority of patients, renal mass biopsy in its current form does not alter clinical management. Its routine use in all-comers is not indicated outside of clinical protocols.

Impact of Ischemia and Procurement Conditions on Gene Expression in Renal Cell Carcinoma

Purpose: Previous studies have shown that ischemia alters gene expression in normal and malignant tissues. There are no studies that evaluated effects of ischemia in renal tumors. This study examines the impact of ischemia and tissue procurement conditions on RNA integrity and gene expression in renal cell carcinoma. Experimental Design: Ten renal tumors were resected without renal hilar clamping from 10 patients with renal clear cell carcinoma. Immediately after tumor resection, a piece of tumor was snap frozen. Remaining tumor samples were stored at 4°C, 22°C, and 37°C and frozen at 5, 30, 60, 120, and 240 minutes. Histopathologic evaluation was conducted on all tissue samples, and only those with greater than 80% tumor were selected for further analysis. RNA integrity was confirmed by electropherograms and quantitated using RNA integrity number index. Altered gene expression was assessed by paired, two-sample t test between the zero time point and aliquots from various conditions obtained from the same tumor. Results: One hundred and forty microarrays were conducted. Some RNA degradation was observed 240 minutes after resection at 37°C. The expression of more than 4,000 genes was significantly altered by ischemia times or storage conditions. The greatest gene expression changes were observed with longer ischemia time and warmer tissue procurement conditions. Conclusion: RNA from kidney cancer remains intact for up to 4 hours post surgical resection regardless of storage conditions. Despite excellent RNA preservation, time after resection and procurement conditions significantly influence gene expression profiles. Meticulous attention to preacquisition variables is of paramount importance for accurate tumor profiling. Clin Cancer Res; 19(1); 42–49. ©2012 AACR.

c-Abl Mediated Tyrosine Phosphorylation of Aha1 Activates Its Co-chaperone Function in Cancer Cells

Summary The ability of Heat Shock Protein 90 (Hsp90) to hydrolyze ATP is essential for its chaperone function. The co-chaperone Aha1 stimulates Hsp90 ATPase activity, tailoring the chaperone function to specific “client” proteins. The intracellular signaling mechanisms directly regulating Aha1 association with Hsp90 remain unknown. Here, we show that c-Abl kinase phosphorylates Y223 in human Aha1 (hAha1), promoting its interaction with Hsp90. This, consequently, results in an increased Hsp90 ATPase activity, enhances Hsp90 interaction with kinase clients, and compromises the chaperoning of non-kinase clients such as glucocorticoid receptor and CFTR. Suggesting a regulatory paradigm, we also find that Y223 phosphorylation leads to ubiquitination and degradation of hAha1 in the proteasome. Finally, pharmacologic inhibition of c-Abl prevents hAha1 interaction with Hsp90, thereby hypersensitizing cancer cells to Hsp90 inhibitors both in vitro and ex vivo.

Mps1 Mediated Phosphorylation of Hsp90 Confers Renal Cell Carcinoma Sensitivity and Selectivity to Hsp90 Inhibitors

SUMMARY The molecular chaperone Hsp90 protects deregulated signaling proteins that are vital for tumor growth and survival. Tumors generally display sensitivity and selectivity toward Hsp90 inhibitors; however, the molecular mechanism underlying this phenotype remains undefined. We report that the mitotic checkpoint kinase Mps1 phosphorylates a conserved threonine residue in the amino-domain of Hsp90. This, in turn, regulates chaperone function by reducing Hsp90 ATPase activity while fostering Hsp90 association with kinase clients, including Mps1. Phosphorylation of Hsp90 is also essential for the mitotic checkpoint because it confers Mps1 stability and activity. We identified Cdc14 as the phosphatase that dephosphorylates Hsp90 and disrupts its interaction with Mps1. This causes Mps1 degradation, thus providing a mechanism for its inactivation. Finally, Hsp90 phosphorylation sensitizes cells to its inhibitors, and elevated Mps1 levels confer renal cell carcinoma selectivity to Hsp90 drugs. Mps1 expression level can potentially serve as a predictive indicator of tumor response to Hsp90 inhibitors.

Compared with radical nephrectomy, nephron-sparing surgery offers a long-term survival advantage in patients between the ages of 20 and 44 years with renal cell carcinomas (≤4 cm): An analysis of the SEER database

PURPOSE Compared with radical nephrectomy (RN), partial nephrectomy (PN) decreases the risk of developing chronic kidney disease. Although numerous studies have demonstrated the survival advantage of PN in older patients, they have been criticized by selection bias toward the procedure owing to comorbidities. We hypothesized that long-standing effects of renal preservation would manifest in a survival advantage of a younger patient population, where selection bias owing to comorbidities is minimized. MATERIALS AND METHODS The Surveillance, Epidemiology, and End Results 18-registries database was queried for patients aged 20 to 44 years surgically treated between 1993 and 2003 for renal cell carcinoma (RCC) ≤ 4 cm with known grade and histology. Patients with prior RCC, multiple tumors, and metastatic or locally advanced disease were excluded. The final cohorts consisted of 222 and 494 subjects treated with PN and RN, respectively. The chi-square and log-rank analyses compared patient and tumor characteristics and patient survival, respectively. RESULTS There were no differences between the groups in demographics or tumor characteristics. Additionally, there was no difference in cancer-specific survival at 5 or 10 years (P = 0.34 and P = 0.1, respectively). Although there was no difference in 5-year overall survival (P = 0.07), PN offered an advantage in 10-year overall survival (P = 0.025). CONCLUSIONS Present Surveillance, Epidemiology, and End Results analyses demonstrate that compared with RN, PN improved overall survival in patients with small, localized RCC. As expected, the survival advantage is observed late and supports the importance of long-term renal functional preservation. Although our study is limited by lack of comorbidities, the results suggest that detrimental effects of RN may have implications on overall survival in younger patients with RCC.

Valproic acid decreases urothelial cancer cell proliferation and induces thrombospondin-1 expression.

BackgroundPrevention of bladder cancer recurrence is a central challenge in the management of this highly prevalent disease. The histone deacetylase inhibitor valproic acid (sodium valproate) has anti-angiogenic properties and has been shown to decrease bladder cancer growth in model systems. We have previously shown reduced expression of thrombospondin-1 in a mouse model and in human bladder cancer relative to normal urothelium. We speculated that inhibition of angiogenesis by valproate might be mediated by this anti-angiogenic protein.MethodsBladder cancer cell lines UMUC3 and T24 were treated with valproate or another histone deacetylase inhibitor, vorinostat, in culture for a period of three days. Proliferation was assessed by alamar blue reduction. Gene expression was evaluated by reverse transcription of RNA and quantitative PCR.ResultsProliferation assays showed treatment with valproate or vorinostat decreased proliferation in both cell lines. Histone deacetylase inhibition also increased relative expression of thrombospondin-1 up to 8 fold at 5 mM valproate.ConclusionsHistone deacetylase inhibitors warrant further study for the prevention or treatment of bladder cancer.

Molecular mechanisms of tissue inhibitor of metalloproteinase 2 in the tumor microenvironment

There has been a recent paradigm shift in the way we target cancer, drawing a greater focus on the role of the tumor microenvironment (TME) in cancer development, progression and metastasis. Within the TME, there is a crosstalk in signaling and communication between the malignant cells and the surrounding extracellular matrix. Matrix metalloproteinases (MMPs) are zinc-dependent endoproteases that have the ability to degrade the matrix surrounding a tumor and mediate tumor growth, angiogenesis and metastatic disease. Their endogenous inhibitors, the Tissue Inhibitors of Metalloproteinases (TIMPs), primarily function to prevent degradation of the ECM via inhibition of MMPs. However, recent studies demonstrate that TIMP family members also possess MMP-independent functions. One TIMP member in particular, TIMP-2, has many distinct properties and functions, that occur independent of MMP inhibition, including the inhibition of tumor growth and reduction of angiogenesis through decreased endothelial cell proliferation and migration. The MMP-independent molecular mechanisms and signaling pathways elicited by TIMP-2 in the TME are described in this review.

Decreasing the indications for radical nephrectomy: a study of multifocal renal cell carcinoma.

Multifocal renal cell carcinoma (RCC) has been reported in 5–25% of cases worldwide. Although management of patients with multifocal RCC has not been clearly defined, presence of multifocal renal masses in one kidney and a normal contralateral kidney has often been considered a reason for performing radical nephrectomy. This study reviews the world literature to provide an accurate estimate of the prevalence of multifocal RCC and evaluates the oncologic outcomes of multifocal RCC after exclusion of patients with known hereditary and familial renal syndromes. A PubMed search of the literature was performed for articles in the English language using the following terms for the query: “multifocal RCC,” “multifocality and RCC,” “multicentric RCC,” or “bilateral RCC.” The references of the published articles were also reviewed for additional publications. Articles that did not specifically exclude patients with familial RCC or known hereditary RCC syndromes were excluded for estimation of multifocality prevalence and oncologic outcomes. After applying our exclusion criteria, nine articles were selected and form the basis of the current analysis. Weighted averages were used to calculate the prevalence of multifocality. Multifocal RCC was found in 6.8% of cases (373 of 5433 patients). Ipsilateral multifocality was found in 6.8% of cases. Bilateral multifocality was found in 11.7% of cases. Of all cases reported in this study, only 10% underwent partial nephrectomy. The rest of the study cohort underwent radical nephrectomy. The review of the literature showed that the use of nephron-sparing techniques in patients with multifocal disease did not compromise oncologic outcomes, despite the need for reoperation in certain cases. In conclusion, multifocal RCC remains a prevalent entity. Most clinicians still prefer to perform radical nephrectomies in these patients despite proven equivalent oncologic outcomes compared to nephron-sparing techniques. Urologists should be aware of these data when proposing treatment options to patients with multifocal RCC.

Argument in favor of performing partial nephrectomy for tumors greater than 7 cm: The metastatic prescription has already been written

The acceptance of partial nephrectomy over the past few decades has been gradual with initial utilization of nephron sparing approach for tumors up to 4 cm and more recently up to 7 cm. The arbitrary cutoff values used in the historic recommendations are based on the oncologic outcomes documenting the increase in metastatic potential of renal lesions that is strongly associated with increase in tumor size. Despite these observations, radical nephrectomy has not been found to be protective from development of metastatic disease, and oncologic outcomes of partial nephrectomy for tumors matched for size or stage have not been inferior to radical nephrectomy. The present manuscript argues for avoidance of specific size cutoffs as patients with larger masses may benefit from maximal preservation of nephrons. These are the very patients at higher risk for metastatic disease, who may benefit from preserved renal function to allow for future additional therapies or adjuvant trials.