FAPα and αSMA mark different subsets of fibroblasts in normal kidney and conventional renal cell carcinoma.

Several studies suggested a correlation between cancer associated fibroblasts (CAF) and cancer progression, but data on conventional renal cell carcinoma (cRCC) is still lacking. We aimed to analyse the impact of αSMA positive myo-CAF and FAPα expressing i-CAF on postoperative relapse of cRCC. We applied immunohistochemistry on tissue-multiarray (TMA) containing 736 consecutively operated cRCC without metastasis at the time of diagnosis. We analysed the correlation between the amount and pattern of αSMA and FAPα expressing CAFs and tumour cells and postoperative tumour relapse. Stromal fibroblasts of each cRCC displayed αSMA immunreaction but only 142 of the 736 tumours showed positive FAPα staining. There was no correlation between the amount of αSMA and or FAPα positive CAFs and tumour progression. However, tumours with large tourtous vessels with strong αSMA positive immunreaction have more then two times higher risk of postoperative tumour relapse (RR=2.198, p = 0.005). Patients with cRCC (57) showing cytoplasmic αSMA staining of tumour cells had a nearly two times higher risk for postoperative progression (RR=1.776, p = 0.014). There is no significant correlation between the density of αSMA or FAPα positive CAFs and postoperative relapse of cRCCs, therefore CAFs in cRCC are not suitable targets for therapy. Further limitation of anti-CAF therapy of cRCC that stromal cells of normal kidney are positive with αSMA antibody.

Src-Family Protein Kinase Inhibitors Suppress MYB Activity in a p300-Dependent Manner.

Recent studies have disclosed transcription factor MYB as a potential drug target for malignancies that are dependent on deregulated MYB function, including acute myeloid leukemia (AML) and adenoid cystic carcinoma (ACC). Although transcription factors are often regarded as undruggable, successful targeting of MYB by low-molecular-weight compounds has recently been demonstrated. In an attempt to repurpose known drugs as novel MYB-inhibitory agents, we have screened libraries of approved drugs and drug-like compounds for molecules with MYB-inhibitory potential. Here, we present initial evidence for the MYB-inhibitory activity of the protein kinase inhibitors bosutinib, PD180970 and PD161570, that we identified in a recent screen. We show that these compounds interfere with the activity of the MYB transactivation domain, apparently by disturbing the ability of MYB to cooperate with the coactivator p300. We show that treatment of the AML cell line HL60 with these compounds triggers the up-regulation of the myeloid differentiation marker CD11b and induces cell death. Importantly, we show that these effects are significantly dampened by forced expression of an activated version of MYB, confirming that the ability to suppress MYB function is a relevant activity of these compounds. Overall, our work identifies several protein kinase inhibitors as novel MYB-inhibitory agents and suggests that the inhibition of MYB function may play a role in their pharmacological impact on leukemic cells.

A synthetic covalent ligand of the C/EBPß transactivation domain inhibits acute myeloid leukemia cells.

C/EBPß has recently emerged as a pro-leukemogenic transcription factor that cooperates with oncoprotein MYB to maintain proliferation and differentiation block of AML cells, making C/EBPß an interesting drug target for AML. Here we have studied the inhibitory potential and biological effects of a synthetic analog of the natural product helenalin, a known inhibitor of C/EBPß. The synthetic compound inhibits C/EBPß by covalent binding to cysteine residues in the transactivation domain, thereby causing up-regulation of differentiation-associated genes, cell death and reduced self-renewal potential of AML cells. Suppression of these effects by ectopic expression of C/EBPß or MYB and gene expression profiling validate C/EBPß as a relevant target of the helenalin-mimic and highlight its role as a pro-leukemogenic factor. Overall, our work demonstrates that the synthetic helenalin mimic acts as a covalent inhibitor of C/EBPß and identifies the cysteine residues in the transactivation domain of C/EBPß as ligandable sites. The helenalin mimic can be considered a potential "lead molecule" but needs further development towards more effective C/EBPß inhibitors before being used as a therapeutic agent.

Characterization of the MYB-inhibitory potential of the Pan-HDAC inhibitor LAQ824.

A large body of work has shown that MYB acts as a master transcription regulator in hematopoietic cells and has pinpointed MYB as a potential drug target for acute myeloid leukemia (AML). Here, we have examined the MYB-inhibitory potential of the HDAC inhibitor LAQ824, which was identified in a screen for novel MYB inhibitors. We show that nanomolar concentrations of LAQ824 and the related HDAC inhibitors vorinostat and panobinostat interfere with MYB function in two ways, by inducing its degradation and inhibiting its activity. Reporter assays show that the inhibition of MYB activity by LAQ824 involves the MYB transactivation domain and the cooperation of MYB with co-activator p300, a key MYB interaction partner and driver of MYB activity. In AML cells, LAQ824-induced degradation of MYB is accompanied by expression of myeloid differentiation markers and apoptotic and necrotic cell death. The ability of LAQ824 to inhibit MYB activity is supported by the observation that down-regulation of direct MYB target genes MYC and GFI1 occurs without apparent decrease of MYB expression already after 2 h of treatment with LAQ824. Furthermore, ectopic expression of an activated version of MYB In HL60 cells counteracts the induction of myeloid differentiation by LAQ824. Overall, our data identify LAQ824 and related HDAC inhibitors as potent MYB-inhibitory agents that exert dual effects on MYB expression and activity in AML cells.

Proteasome inhibitors suppress MYB oncogenic activity in a p300-dependent manner.

Studies of the role of MYB in human malignancies have highlighted MYB as a potential drug target for acute myeloid leukemia (AML) and adenoid cystic carcinoma (ACC). Although transcription factors are often considered un-druggable, recent work has demonstrated successful targeting of MYB by low molecular weight compounds. This has fueled the notion that inhibition of MYB has potential as a therapeutic approach against MYB-driven malignancies. Here, we have used a MYB reporter cell line to screen a library of FDA-approved drugs for novel MYB inhibitors. We demonstrate that proteasome inhibitors have significant MYB-inhibitory activity, prompting us to characterize the proteasome inhibitor oprozomib in more detail. Oprozomib was shown to interfere with the ability of the co-activator p300 to stimulate MYB activity and to exert anti-proliferative effects on human AML and ACC cells. Overall, our work demonstrated suppression of oncogenic MYB activity as a novel result of proteasome inhibition.

C/EBPß is a MYB- and p300-cooperating pro-leukemogenic factor and promising drug target in acute myeloid leukemia.

Transcription factor MYB has recently emerged as a promising drug target for the treatment of acute myeloid leukemia (AML). Here, we have characterized a group of natural sesquiterpene lactones (STLs), previously shown to suppress MYB activity, for their potential to decrease AML cell proliferation. Unlike what was initially thought, these compounds inhibit MYB indirectly via its cooperation partner C/EBPß. C/EBPß-inhibitory STLs affect the expression of a large number of MYB-regulated genes, suggesting that the cooperation of MYB and C/EBPß broadly shapes the transcriptional program of AML cells. We show that expression of GFI1, a direct MYB target gene, is controlled cooperatively by MYB, C/EBPß, and co-activator p300, and is down-regulated by C/EBPß-inhibitory STLs, exemplifying that they target the activity of composite MYB-C/EBPß-p300 transcriptional modules. Ectopic expression of GFI1, a zinc-finger protein that is required for the maintenance of hematopoietic stem and progenitor cells, partially abrogated STL-induced myelomonocytic differentiation, implicating GFI1 as a relevant target of C/EBPß-inhibitory STLs. Overall, our data identify C/EBPß as a pro-leukemogenic factor in AML and suggest that targeting of C/EBPß may have therapeutic potential against AML.

Bcr-TMP, a Novel Nanomolar-Active Compound That Exhibits Both MYB- and Microtubule-Inhibitory Activity.

Studies of the role of MYB in human malignancies have highlighted MYB as a potential drug target for acute myeloid leukemia (AML) and adenoid cystic carcinoma (ACC). Here, we present the initial characterization of 2-amino-4-(3,4,5-trimethoxyphenyl)-4H-naphtho[1,2-b]pyran-3-carbonitrile (Bcr-TMP), a nanomolar-active MYB-inhibitory compound identified in a screen for novel MYB inhibitors. Bcr-TMP affects MYB function in a dual manner by inducing its degradation and suppressing its transactivation potential by disrupting its cooperation with co-activator p300. Bcr-TMP also interferes with the p300-dependent stimulation of C/EBPß, a transcription factor co-operating with MYB in myeloid cells, indicating that Bcr-TMP is a p300-inhibitor. Bcr-TMP reduces the viability of AML cell lines at nanomolar concentrations and induces cell-death and expression of myeloid differentiation markers. It also down-regulates the expression of MYB target genes and exerts stronger anti-proliferative effects on MYB-addicted primary murine AML cells and patient-derived ACC cells than on their non-oncogenic counterparts. Surprisingly, we observed that Bcr-TMP also has microtubule-disrupting activity, pointing to a possible link between MYB-activity and microtubule stability. Overall, Bcr-TMP is a highly potent multifunctional MYB-inhibitory agent that warrants further investigation of its therapeutic potential and mechanism(s) of action.

The Role of Genetic Analysis in Correct Diagnosis of Eosinophilic Variant of Chromophobe Renal Cell Carcinoma.

BACKGROUND/AIM: It has been suggested that eosinophilic variant of chromophobe renal cell carcinoma (chRCC) with low chromosome number or lack of genomic alteration has an excellent prognosis in comparison to classic chRCC. The aim of our study was to analyse the phenotypical variations of 77 chRCCs, including 7 eosinophilic ones, each diagnosed unequivocally by genetic means. MATERIALS AND METHODS: DNA isolated from chRCCs was subjected to array comparative genomic hybridisation (CGH) for establishing the chromosome alteration. Original histological slides were evaluated for cellular phenotype and growth pattern and compared to the genetic alterations. RESULTS: Loss of the entire chromosome 1, 2, 6, 10, 13, 17 and 21 occurred in 95%, 94%, 86% 90% 82% 90% and 66% of the cases, respectively. The number of chromosome alterations in eosinophilic forms of chRCC corresponded to those found in classic chRCC with pale-reticular cytoplasm or mixed cellular characteristics. Three of seven eosinophilic variants with loss of 4, 10 and 11 chromosomes showed metastasis at the time of diagnosis whereas only 3 metastatic tumors were noticed among the 70 classic chRCC. We did not find discriminating difference in number of chromosome alteration between classic and eosinophilic forms of chRCC. CONCLUSION: Eosinophilic chRCC has a more aggressive biology than the classic form. To avoid diagnostic pitfall of eosinophilic renal cell tumors with uncertain diagnosis, a genetic analysis should be carried out.

Cytoplasmic Expression of AXL Is Associated With High Risk of Postoperative Relapse of Conventional Renal Cell Carcinoma.

BACKGROUND/AIM: Despite early detection by widespread use of abdominal imaging more than 40% of patients with conventional renal cell carcinoma (RCC) will die due to metastatic disease. Small kinase inhibitors for AXL receptor tyrosine kinase may delay the progression of metastatic cRCC. PATIENTS AND METHODS: We analysed AXL expression by immunohistochemistry on tissue multi arrays of 691 conventional RCC without metastasis at the time of nephrectomy. RESULTS: The Kaplan-Meier survival analysis indicated a poor disease-specific survival rates for patients with tumour showing cytoplasmic AXL staining, whereas expression on the cell membrane is associated with excellent disease outcome. Multivariate Cox regression analysis identified cytoplasmic AXL expression as an independent prognostic factor indicating a five-times higher risk of postoperative tumour progression (RR=5.048; 95% CI=2.391-10.657; p<0.001). CONCLUSION: Detecting cytoplasmic expression of AXL can be used to define a subset of conventional RCC with high risk of progression, thus identifying patients for more aggressive surveillance and adjuvant AXL inhibitor treatment as early as possible.

Expression of RARRES1 and AGBL2 and progression of conventional renal cell carcinoma.

BACKGROUND: Approximately 15% of clinically localised conventional renal cell carcinoma (RCC) will develop metastasis within 5 years of follow-up. The aim of this study was to identify biomarkers predicting the postoperative tumour relapse. METHODS: Tissue microarrays of conventional RCC from a cohort of 691 patients without metastasis at the time of operation were analysed by immunohistochemistry for the expression of carboxypeptase inhibitor RARRES1 and its substrate carboxypeptidase AGBL2. Univariate and multivariate Cox regression models were addressed to postoperative tumour relapse and the metastasis-free survival time was estimated by Kaplan-Meier analysis. RESULTS: In multivariate analysis, the lack of staining or cytoplasmic staining of RARRES1 was a significant risk factor indicating five times higher risk of cancer relapse. Combining its co-expression with AGBL2, we found that RARRES1 cytoplasmic/negative and AGBL2-positive/negative staining is a significant risk factor for tumour progression indicating 11-15 times higher risk of cancer relapse, whereas the membranous RARRES1 expression, especially its co-expression with AGBL2, associated with excellent disease outcome. CONCLUSIONS: RARRES1 and AGBL2 expression defines groups of patients at low and high risk of tumour progression and may direct an active surveillance to detect metastasis as early as possible and to apply adjuvant therapy.

Monensin, a novel potent MYB inhibitor, suppresses proliferation of acute myeloid leukemia and adenoid cystic carcinoma cells.

The master transcriptional regulator MYB is a key oncogenic driver in several human neoplasms, particularly in acute myeloid leukemia (AML) and adenoid cystic carcinoma (ACC). MYB is therefore an attractive target for drug development in MYB-activated malignancies. Here, we employed a MYB-reporter cell line and identified the polyether ionophores monensin, salinomycin, and nigericin as novel inhibitors of MYB activity. As a proof of principle, we show that monensin affects the expression of a significant number of MYB-regulated genes in AML cells and causes down-regulation of MYB expression, loss of cell viability, and induction of differentiation and apoptosis. Furthermore, monensin significantly inhibits proliferation of primary murine AML cells but not of normal hematopoietic progenitors, reflecting a high MYB-dependence of leukemic cells and underscoring the efficacy of monensin in MYB-activated malignancies. Importantly, monensin also suppressed the viability and non-adherent growth of adenoid cystic carcinoma (ACC) cells expressing MYB-NFIB fusion oncoproteins. Our data show that a single compound with significant MYB-inhibitory activity is effective against malignant cells from two distinct MYB-driven human neoplasms. Hence, monensin and related compounds are promising molecular scaffolds for development of novel MYB inhibitors.

M2 Macrophage Marker Chitinase 3-Like 2 (CHI3L2) Associates With Progression of Conventional Renal Cell Carcinoma.

BACKGROUND/AIM: In spite of early detection, appoximately 15% of the small renal cell carcinomas (RCC) will develop metastasis within 5 years follow-up. The aim of this study was to identify new biomarkers to estimate the postoperative relapse of the most common conventional RCC. PATIENTS AND METHODS: Tissue multi arrays of conventional RCC without metastasis at the time of operation from a cohort of 634 patients were analysed by immunohistochemistry for expression of the chitinase 3-like protein 2 (CHI3L2). Cancer specific survival of patients was estimated with Kaplan-Meier analysis, univariate and multivariate Cox regression models. RESULTS: Kaplan-Meier analysis estimated a shorter cancer-free survival for patients with CHI3L2 positive tumors. In multivariate analysis, the CHI3L2 positivity associated with a 3.5 times higher risk for tumor relapse (p<0.001). CONCLUSION: Expression of CHI3L2 in tumor cells of conventional RCC define a group of patients at high risk for postoperative progression.

Dual role of KRT17: development of papillary renal cell tumor and progression of conventional renal cell carcinoma.

Expression of KRT17 has been described in multi-layered epithelia as well as in tumors derived from these cells. In cancers arising from KRT17 negative single layered epithelia neo-expression of KRT17 has been associated with tumor progression. To obtain more insight into the biology of kidney cancers we have investigated KRT17 expression by immunohistochemistry in normal kidney, in papillary preneoplastic lesions and in 151 papillary and 692 conventional renal cell carcinomas placed on tissue microarray. We found a positive staining in ureteric bud and collecting duct cells in foetal kidney, in all papillary preneoplastic lesions and also in 77% of the 151 papillary renal cell tumors indicating a continuos KRT17 expression during tumor development. The neo-expression of KRT17 in conventional renal cell carcinomas, which derives from KRT17 negative proximal tubules showed a significant correlation with postoperative tumor relapse (RR=2.50; 95% CI=1.59-3.94; p<0.001). In conclusion, the continuous expression of KRT17 from emerging fetal kidney tubules and microscopic pre-neoplastic lesions towards papillary renal cell tumors and its neo-expression in aggressive growing conventional renal cell carcinomas reflects the multiple function of KRT17 in kidney cancers with distinct natural history. This should be taken into account in clinical managements and therapy.

IL6 Shapes an Inflammatory Microenvironment and Triggers the Development of Unique Types of Cancer in End-stage Kidney.

BACKGROUND/AIM: Chronic inflammation in end-stage kidney is associated with the development of pre-neoplastic lesions and renal cell tumors. The aim of this study was to clarify the role of the inflammatory microenvironment in this process. MATERIALS AND METHODS: We used representative microscopic slides from 11 end stage-kidneys containing pre-neoplastic lesions and tumors and applied immunohistochemistry to detect IL-6, SAA1 and LBP expression. We also applied array-based comparative genomic hybridization (CGH) analysis to detect genomic changes in tumor cells. RESULTS: We identified strong expression of IL6, LBP and SAA1 in activated stromal fibroblasts, in proliferating epithelial and tumor cells. Array CGH detected unusual genomic changes in tumor cells. CONCLUSION: Our data indicate that expression of IL6, acute phase protein SAA1 and LBP maintain a long-lasting inflammatory microenvironment that leads to remodeling of end-stage kidneys and the development of unique types of renal cell tumors.

Genomic profiling of papillary renal cell tumours identifies small regions of DNA alterations: a possible role of HNF1B in tumour development.

AIMS: Papillary renal cell tumours (RCT) are characterized by specific trisomies. The aim of this study was to identify small regions of duplication marking putative tumour genes. METHODS AND RESULTS: Full-tiling path bacterial artificial chromosome (BAC) array hybridization of 20 papillary RCTs confirmed the duplication of chromosomes 7 and 17 and also displayed smaller regions of gains/amplifications of 1.3-13.1 Mb in size. Detailed analysis showed a microamplification of BAC clones containing the MET at the 7q31.2 and also amplification of a DNA segment harbouring the transcription factor hepatocyte nuclear factor 1 beta (HNF1B) at chromosome 17q12. Nuclear expression of HNF1B protein was detected in 38 of 67 papillary RCTs, in five of five mucinous tubular and spindle cell carcinomas (MTSCC) and five of five metanephric adenomas by immunohistochemistry. Moreover, nine nephrogenic rests containing tubular differentiated structures and all 14 and five precursor lesions associated with papillary RCTs and MTSCCs, respectively, showed strong nuclear positivity when compared to the expression level in proximal tubules of the corresponding normal kidney. CONCLUSIONS: Our findings indicate a role of HNF1B in association with the high frequency of chromosome 17q duplication in the development of papillary RCTs and MTSCCs as well as in their precursor lesions.

Lack of KISS1R expression is associated with rapid progression of conventional renal cell carcinomas.

The mortality of patients with conventional renal cell carcinomas (RCC) correlates directly with the development of metastasis, which cannot be reliably predicted simply by TNM classification. The aim of this study was to identify genes associated with the tumour progression. We have analysed the global gene expression in conventional RCCs, including those with and without progression by Affymetrix GeneChip and selected the genes by gene set enrichment analysis. The expression and function of KISS1R was validated by RT-PCR, western blotting and immunohistochemistry and by in vitro experiments. An immunohistochemical and clinical follow-up study showed that lack of KISS1R expression is associated with rapid progression of tumours. In vitro studies showed that activation of KISS1/KISS1R signalling by kisspeptin treatment decreases the motility and invasive capacity of tumour cells. The kisspeptin treatment also induces the expression of KISS1R in tumour cells in vitro and activates signalling in cases without constitutional expression of the receptor. Expression of the KISS1R protein can be used for estimating the prognosis of conventional RCCs. Confirming the activation of KISS1R signalling in vivo may open a way for kisspeptin treatment of patients with conventional RCCs.

Molecular pathology of chromophobe renal cell carcinoma: a review.

The recognition of chromophobe renal cell carcinoma (RCC) among other distinct types of renal cell tumors (RCT) based on light-microscopic features, such as cytoplasmic and nuclear characteristics, might pose a dilemma in some cases because of morphological pattern overlapping with renal oncocytoma or conventional RCC. The present article reviews chromophobe RCC with focus on aspects of its molecular pathology, which was shown using ancillary modern microarray-based technology that can distinguish it from its mimics and therefore be helpful for its correct diagnosis. Although the high resolution DNA-microarray analyses excluded with all certainty the occurrence of small specific alterations, the loss of entire chromosomes 2, 10, 13, 17 and 21 occurs exclusively in chromophobe RCC and therefore probes localized at these chromosomes might be used to establish the diagnosis of chromophobe RCC in cases with uncertain histology. The usefulness of proposed candidate genes selected by the global gene expression analyses in the diagnostic pathology is far below expectations. The conflicting staining patterns, together with the poor specificity of used antibodies, leads us to believe that these candidate immunomarkers might not help in the separation of chromophobe RCC, with the exception of CD82, which has recently been suggested to be used for routine histological diagnosis.

Molecular pathology of renal oncocytoma: a review.

Differentiating renal oncocytoma from its renal cell carcinoma (RCC) mimics, particularly chromophobe RCC, can be difficult, especially when limited tissue is available for evaluation and requires sophisticated microscopic, ultrastructural and immunohistochemical evaluation. In this review, the relevant literature has been reviewed, and supporting data obtained by applying modern microarray-based technologies are discussed with a focus on molecular pathology of renal oncocytoma. The high resolution whole-genome DNA-microarray based analyses excluded with all certainty the occurrence of small specific alterations. Renal oncocytomas are characterized by variable chromosomal patterns. The number of genes selected by global gene expression analyses and their usefulness in the diagnostic pathology based on immunohistochemical evaluation is far below the expectations. The conflicting staining patterns, together with the poor specificity of proposed antibodies, leads us to believe that these candidate immunomarkers might not help in the separation of these tumors. Applying DNA based tools might help in the diagnosis of renal oncocytoma with uncertain histology. However, only the combination of all available techniques could give reliable information.

Molecular analysis of germline t(3;6) and t(3;12) associated with conventional renal cell carcinomas indicates their rate-limiting role and supports the three-hit model of carcinogenesis.

We describe the molecular analysis of chromosomal rearrangements in familial t(3;6)(p12.3;q24.3) and t(3;12)(q13.13;q24.23) associated with the development of conventional renal cell carcinomas (RCC). We mapped the breakpoints by high-density oligo array comparative genomic hybridization of tumor cells in t(3;6) at chromosome 3p12.3 between PDZRN3 and CNTN3; the chromosomal rearrangement at 6q24.3 was mapped within the seventh intron of the STXBP5 gene. In the second case, the break at 3q13.13 was mapped downstream of PVRL3 and the breakpoint at 12q24.23 between HSPB8 and CCDC60, one allele of the latter being deleted. Reverse transcriptase polymerase chain reaction analysis of the PDZRN3, CNTN3, STXBP5, PVRL3, HSPB8, and CCDC60 genes revealed slight variation in the copy number of transcripts, but without correlation to the chromosomal rearrangements in translocation-associated and sporadic conventional RCCs. Loss of heterozygosity at chromosome 3p and mutation of VHL occurred at the same frequency in both familial and sporadic cases. Based on our model of nonhomologous chromatid exchange and the data on molecular studies, we suggest that the germline translocation serves as a rate-limiting step toward tumor development by generating a high number of cells with loss of the derivative chromosome carrying the 3p segment.

Oligoarray comparative genomic hybridization of renal cell tumors that developed in patients with acquired cystic renal disease.

Renal cell carcinoma occurs at higher frequency in acquired cystic renal disease than in the general population. We have analyzed 4 tumors obtained from the kidneys of 2 patients with acquired cystic renal disease, including 2 conventional renal cell carcinomas and 2 acquired cystic renal disease-associated tumors, for genetic alterations. DNA changes were established by applying the 44K Agilent Oligonucleotide Array-Based CGH (Agilent Technologies, Waldbronn, Germany), and mutation of VHL gene was detected by direct sequencing of the tumor genome. DNA losses and mutation of the VHL gene, which are characteristic for conventional renal cell carcinomas, were seen in 2 of the tumors. The acquired cystic renal disease-associated eosinophilic-vacuolated cell tumor showed gain of chromosomes 3 and 16. No DNA alterations occurred in the papillary clear cell tumor. We suggest that not only the morphology but also the genetics of renal cell tumors associated with acquired cystic renal disease may differ from those occurring in the general population.