Resistance to sunitinib in renal cell carcinoma: From molecular mechanisms to predictive markers and future perspectives.

The introduction of agents that inhibit tumor angiogenesis by targeting vascular endothelial growth factor (VEGF) signaling has made a significant impact on the survival of patients with metastasized renal cell carcinoma (RCC). Sunitinib, a tyrosine kinase inhibitor of the VEGF receptor, has become the mainstay of treatment for these patients. Although treatment with sunitinib substantially improved patient outcome, the initial success is overshadowed by the occurrence of resistance. The mechanisms of resistance are poorly understood. Insight into the molecular mechanisms of resistance will help to better understand the biology of RCC and can ultimately aid the development of more effective therapies for patients with this infaust disease. In this review we comprehensively discuss molecular mechanisms of resistance to sunitinib and the involved biological processes, summarize potential biomarkers that predict response and resistance to treatment with sunitinib, and elaborate on future perspectives in the treatment of metastasized RCC.

[Novel prognostic marker in invasive breast cancer. ITIH5 expression is abrogated by aberrant promoter methylation]. / Neuer Prognosemarker beim invasiven Mammakarzinom. ITIH5-Expression wird über aberrante Promotormethylierung inaktiviert.

We have recently characterized ITIH5 as a new extracellular matrix protein that exhibits clear expression loss in a variety of human tumour entities, including breast cancer. The aim of the present study was to decipher the molecular cause of ITIH5 expression loss in breast cancer and to learn more about the possible role of this molecule in cancer diseases. ITIH5 protein expression was found to be strongly reduced in 42% of invasive breast carcinomas-interestingly, with significant association with poor patient outcome. ITIH5 promoter methylation was frequently detected in breast cell lines and in primary carcinomas (40%), and it was functionally correlated with loss of ITIH5 mRNA expression. Moreover, ITIH5 promoter methylation was also significantly associated with poor clinical patient outcome and also with the occurrence of lymph node and distant metastases. In conclusion, we propose that ITIH5 may represent a novel metastasis repressor in human breast cancer. Both ITIH5 protein expression and ITIH5 promoter methylation may serve as prognostic biomarkers, thereby helping improve clinical patient outcome.

[Promoter methylation of ID4. A marker for recurrence-free survival in human breast cancer]. / Promotormethylierung von ID4. Ein Marker für die Rezidivbildung des humanen Mammakarzinoms.

The aim of this study was to unravel the role of the transcription factor inhibitor of DNA binding 4 (ID4) in human breast carcinogenesis in more detail, especially the impact of ID4 promoter methylation on disease progression. Demethylating treatment of breast cancer cell lines was associated with ID4 reexpression. ID4 promoter methylation was frequently observed in primary breast cancer samples (68.9%, 117/170). We found a very tight correlation (p<0.001) between ID4 promoter methylation and loss of ID4 mRNA expression in these specimens. For breast tissue as the first tumour entity analyzed in detail, we could show a direct correlation between ID4 promoter methylation and loss of ID4 expression on both the mRNA and protein level. Interestingly, ID4 promoter methylation was a factor for unfavourable recurrence-free survival (p=0.036) and increased the patient's risk for lymph node metastases (p=0.030). Our data suggest that ID4 is a potential tumour suppressor gene in human breast tissues that undergoes epigenetic silencing during carcinogenesis, leading to an increased risk for tumour relapse. Thus, ID4 methylation status could serve as a prognostic biomarker in human breast cancer.

The extracellular matrix protein ITIH5 is a novel prognostic marker in invasive node-negative breast cancer and its aberrant expression is caused by promoter hypermethylation.

Inter-alpha-trypsin inhibitors (ITIs) are protease inhibitors stabilizing the extracellular matrix. ITIs consist of one light (bikunin) and two heavy chains (ITIHs). We have recently characterized ITIH5, a novel member of the ITIH gene family, and showed that its messenger RNA is lost in a high proportion of breast tumours. In the present study, an ITIH5-specific polyclonal antibody was generated, validated with western blot and used for immunohistochemical analysis on a tissue microarray; ITIH5 was strongly expressed in epithelial cells of normal breast (n=11/15), while it was lost or strongly reduced in 42% (92/217) of invasive breast cancers. ITIH5 expression in invasive carcinomas was associated with positive expression of oestrogen receptor (P=0.008) and histological grade (P=0.024). Correlation of ITIH5 expression with clinical outcome revealed that patients with primary tumours retaining abundant ITIH5 expression had longer recurrence-free survival (RFS; P=0.037) and overall survival (OS; P=0.044), compared to those with reduced expression (mean RFS: 102 vs 78 months; mean OS: 120 vs 105 months). Methylation-specific PCR analysis frequently showed strong methylation of the ITIH5 promoter in primary breast tumours (41%, n=109) and breast cancer cell lines (n=6). Methylation was significantly associated with mRNA loss (P<0.001; n=39), and ITIH5 expression was induced after treatment of tumour cell lines with the demethylating agent 5-aza-2'-deoxycytidine. Moreover, ITIH5 promoter methylation was significantly associated with reduced OS (P=0.008). The cellular function of ITIH5 was evaluated by forced expression of a full-length ITIH5 complementary DNA in the breast cancer cell line MDA-MB-231, which does not endogenously express ITIH5. ITIH5-expressing clones showed a 40% reduced proliferation rate compared to mock-transfected cells. Overall, these data show that promoter methylation-mediated loss of ITIH5 expression is associated with unfavourable outcome in breast cancer patients, and thus ITIH5 could be used as a prognostic marker, although this marker is not multivariate independent due to its close association with ER expression. Our data indicate that ITIH5 is a candidate class II tumour suppressor gene and could be involved in tumour progression, invasion and metastasis, as its absence is associated with increased proliferation rates and a prognostic value indicating poor clinical outcome.

Frequent loss of SFRP1 expression in multiple human solid tumours: association with aberrant promoter methylation in renal cell carcinoma.

Oncogenic wingless-related mouse mammary tumour virus (Wnt) signalling, caused by epigenetic inactivation of specific pathway regulators like the putative tumour suppressor secreted frizzled-related protein 1 (SFRP1), may be causally involved in the carcinogenesis of many human solid tumours including breast, colon and kidney cancer. To evaluate the incidence of SFRP1 deficiency in human tumours, we performed a large-scale SFRP1 expression analysis using immunohistochemistry on a comprehensive tissue microarray (TMA) comprising 3448 tumours from 36 organs. This TMA contained 132 different tumour subtypes as well as 26 different normal tissues. Although tumour precursor stages of, for example kidney, colon, endometrium or adrenal gland still exhibited moderate to abundant SFRP1 expression, this expression was frequently lost in the corresponding genuine tumours. We defined nine novel tumour entities with apparent loss of SFRP1 expression, i.e., cancers of the kidney, stomach, small intestine, pancreas, parathyroid, adrenal gland, gall bladder, endometrium and testis. Renal cell carcinoma (RCC) exhibited the highest frequency of SFRP1 loss (89% on mRNA level; 75% on protein level) and was selected for further analysis to investigate the cause of SFRP1 loss in human tumours. We performed expression, mutation and methylation analysis in RCC and their matching normal kidney tissues. SFRP1 promoter methylation was frequently found in RCC (68%, n=38) and was correlated with loss of SFRP1 mRNA expression (p<0.05). Although loss of heterozygosity was found in 16% of RCC, structural mutations in the coding or promoter region of the SFRP1 gene were not observed. Our results indicate that loss of SFRP1 expression is a very common event in human cancer, arguing for a fundamental role of aberrant Wnt signalling in the development of solid tumours. In RCC, promoter hypermethylation seems to be the predominant mechanism of SFRP1 gene silencing and may contribute to initiation and progression of this disease.

Aberrant methylation of the Wnt antagonist SFRP1 in breast cancer is associated with unfavourable prognosis.

The canonical Wnt signalling pathway plays a key role during embryogenesis and defects in this pathway have been implicated in the pathogenesis of various types of tumours, including breast cancer. The gene for secreted frizzled-related protein 1 (SFRP1) encodes a soluble Wnt antagonist and is located in a chromosomal region (8p22-p12) that is often deleted in breast cancer. In colon, lung, bladder and ovarian cancer SFRP1 expression is frequently inactivated by promoter methylation. We have previously shown that loss of SFRP1 protein expression is a common event in breast tumours that is associated with poor overall survival in patients with early breast cancer. To investigate the cause of SFRP1 loss in breast cancer, we performed mutation, methylation and expression analysis in human primary breast tumours and breast cell lines. No SFRP1 gene mutations were detected. However, promoter methylation of SFRP1 was frequently observed in both primary breast cancer (61%, n=130) and cell lines analysed by methylation-specific polymerase chain reaction (MSP). We found a tight correlation (P<0.001) between methylation and loss of SFRP1 expression in primary breast cancer tissue. SFRP1 expression was restored after treatment of tumour cell lines with the demethylating agent 5-aza-2'-deoxycytidine. Most interestingly, SFRP1 promoter methylation was an independent factor for adverse patient survival in Kaplan-Meier analysis. Our results indicate that promoter hypermethylation is the predominant mechanism of SFRP1 gene silencing in human breast cancer and that SFRP1 gene inactivation in breast cancer is associated with unfavourable prognosis.

[Epigenetic inactivation of the WNT antagonist SFRP1 in breast cancer]. / Epigenetische Inaktivierung des WNT-Antagonisten SFRP1 im Mammakarzinom.

The WNT signalling pathway plays a central role during embryonic development of multi-cellular organisms, especially for the temporal and spatial specification of organs (e.g. WNT4 in kidney development), a process called pattern formation. Interestingly, genes of the WNT pathway are deregulated in a variety of solid tumours, being considerably up- or down-regulated compared to their expression in the corresponding normal tissue. Some members like WNT1 have demonstrated oncogenic properties in animal models. The SFRP1 gene on chromosome 8 p12 is a negative regulator of the WNT pathway. SFRP1 protein is supposed to bind WNT1 molecules thereby inhibiting the activation of frizzled receptors and the WNT pathway. Characterising an SFRP1-specific antibody we could show that loss of SFRP1 is an extremely common event in breast cancer, i.e. SFRP1 was considerably down-regulated in 73% (n = 1967) of analysed invasive breast cancers. SFRP1 loss is associated with unfavourable prognosis in early breast cancer (pT1 tumours). To analyse the cause of SFRP1 inactivation in breast cancer we performed a parallel expression and promoter methylation analysis in human breast cancer and tumour cell lines. RT-PCR techniques and methylation-specific PCR (MSP) were applied. All tumorigenic cell lines analysed exhibited complete promoter methylation and did not express detectable amounts of SFRP1 mRNA. SFRP1 expression could be restored in these cell lines after treatment with 5-Aza-2'-deoxycytidine, a demethylating agent. Human primary breast cancer was methylated in nearly 75% of cases. Our results indicate that epigenetic inactivation by methylation is the predominant mechanism of SFRP1 gene silencing in breast cancer.

Biosynthesis of phytochelatins in the fission yeast. Phytochelatin synthesis: a second role for the glutathione synthetase gene of Schizosaccharomyces pombe.

By complementation screening of a cadmium-sensitive Schizosaccharomyces pombe mutant deficient in phytochelatin synthesis, but with 44% of the wild-type glutathione content, we cloned a DNA fragment involved in phytochelatin synthesis. Sequence analysis revealed that it encodes the second enzyme involved in glutathione (GSH) biosynthesis, glutathione synthetase (GSH2) (E.C.6.3.2.3, Wang and Oliver, 1997). The mutant allele shows a single base-pair exchange at the 3' end of the reading frame leading to a single amino acid change from glycine to aspartate. This mutation leads to a significant reduction of phytochelatin synthesis, whereas glutathione synthesis is impaired to a far lesser extent. Complementation with the Arabidopsis thaliana GSH2 cDNA led to a partial restoration of phytochelatin synthesis. These data strongly suggest that the GSH2 gene encodes a bifunctional enzyme that is able to catalyse both the synthesis of GSH by adding glycine to the dipeptide (gammaGlu-Cys) and the synthesis of phytochelatins. The sequence has been submitted to EMBL, Accession No. Y08414.