Rainer Engers

Prevalence and Heterogeneity of KRAS, BRAF, and PIK3CA Mutations in Primary Colorectal Adenocarcinomas and Their Corresponding Metastases

Purpose: Epidermal growth factor receptor (EGFR) antibody therapy is established in patients with wild-type KRAS colorectal carcinoma; however, up to 50% of these patients do not respond to this therapy. To identify the possible causes of this therapy failure, we searched for mutations in different EGFR-dependent signaling proteins and analyzed their distribution patterns in primary tumors and corresponding metastases. Experimental Design: Tumor tissues, macrodissected from tumor centers, invasion fronts (n = 100), lymph nodes (n = 55), and distant metastases (n = 20), respectively, were subjected to DNA extraction and mutation analysis of KRAS, BRAF, and PIK3CA. Results: Activating mutations were detected in 41% (KRAS), 7% (BRAF), and 21% (PIK3CA) of the primary tumors. By comparing tumor centers and invasion fronts, the intratumoral heterogeneity of KRAS, BRAF, and PIK3CA mutations was observed in 8%, 1%, and 5% of primary tumors, respectively. Heterogeneity between primary tumors and lymph node metastases was found in 31% (KRAS), 4% (BRAF), and 13% (PIK3CA) of the cases. Heterogeneity between primary tumors and distant metastases was present in two patients (10%) for KRAS and one patient for PIK3CA (5%), but not for BRAF. Discordant results between primary tumors and metastases could markedly be reduced by testing the additional tumor samples. Conclusions: Failure of EGFR antibody therapy in patients with wild-type KRAS colorectal cancer may result from activating BRAF or PIK3CA mutations and false-negative sequencing results caused by intratumoral heterogeneity. Due to the particularly high rates of heterogeneity between primary tumors and lymph node metastases, the latter are least suitable for diagnostic mutation analysis. Clin Cancer Res; 16(3); 790–9

Genomewide DNA hypomethylation is associated with alterations on chromosome 8 in prostate carcinoma.

To elucidate the relationship between genomewide DNA hypomethylation and chromosome instability, 55 prostate carcinoma specimens were analyzed for extent of hypomethylation by Southern blot analysis of LINE‐1 sequence methylation and for loss or gain of chromosomal material by comparative genomic hybridization. Seventeen (31%) tumors showed strong hypomethylation of DNA, whereas four (7%) displayed slight hypomethylation and the rest of the tumors normal‐level methylation. Chromosomal aberrations were observed in 34 carcinomas. The most frequent chromosomal alterations were loss of 13q in 18 cases and aberrations in 8p (loss) or 8q (gain) in 16 cases. The presence of chromosomal loss or gain was significantly associated with the presence of strong hypomethylation. A striking correlation (P = 0.00001) was observed between aberrations on chromosome 8 and hypomethylation, whereas no association was seen between DNA hypomethylation and loss of 13q. The association between DNA hypomethylation and the presence of metastases was statistically significant (P = 0.044), and both chromosomal alterations and DNA hypomethylation tended to be more frequent in higher‐stage tumors. In conclusion, the data indicate that hypomethylation is associated with chromosomal instability in prostate cancer. Specifically, a surprisingly strong association between alterations on chromosome 8 and genomewide hypomethylation was found. This association suggests that DNA hypomethylation and alterations in chromosome 8 may be mechanistically linked to each other in prostate carcinoma.

Mechanisms of tumor metastasis: cell biological aspects and clinical implications.

Abstract The clinical course – and hence the prognosis – of patients suffering from malignant tumors are essentially determined by the capability of tumor cells to metastasize. During the past decade knowledge about genetic aberrations, as well as molecular and cell biological mechanisms which are involved in the regulation of tumor metastasis, has dramatically increased and consequently led to the development of new theoretical and experimental strategies in cancer treatment. The objective of this review is not only to give an overview about the principal cell biological and molecular mechanisms of tumor metastasis, but also to discuss potential therapeutical options resulting from this knowledge.

The Rac Activator Tiam1 Is a Wnt-responsive Gene That Modifies Intestinal Tumor Development

Mutations in the canonical Wnt signaling pathway leading to its activation are known to cause the majority of intestinal tumors. However, few genes targeted by this pathway have been demonstrated to affect tumor development in vivo. Here we show that Tiam1, a selective Rac GTPase activator, is a Wnt-responsive gene expressed in the base of intestinal crypts and up-regulated in mouse intestinal tumors and human colon adenomas. Moreover, by comparing tumor development in APC mutant Min (multiple intestinal neoplasia) mice expressing or lacking Tiam1, we found that Tiam1 deficiency significantly reduces the formation and growth of polyps in vivo. However, invasion of malignant intestinal tumors is enhanced by a lack of Tiam1. In line with this, knock-down of Tiam1 reduced the growth potential of human colorectal cancer cells and their ability to form E-cadherin-based adhesions, a prerequisite for local invasion of tumor cells. Our data indicate a novel cross-talk between Tiam1-Rac and canonical Wnt-signaling pathways that influences intestinal tumor formation and progression.

Tiam1 mutations in human renal-cell carcinomas.

Tiam1 activates the Rho‐like GTPase Rac1, and studies indicate that Tiam1–Rac1 signaling affects invasion in different ways depending on the cell type studied. However, no investigations on Tiam1 in human tumors have been reported. Here, we show that for 4 of 5 human renal‐cell carcinoma (RCC) cell lines the expression levels of Tiam1 tended to be inversely correlated with in vitro invasiveness, whereas no obvious correlation could be found between the expression levels of Rac1 and invasion. Subsequent mutation analysis of these cell lines revealed no mutations in Rac1 but up to 5 different point mutations in the Tiam1 gene. Of these, 1 mutation (A441G) was located in the NH2‐terminal pleckstrin homology domain, which is essential for membrane localization and functional activity of Tiam1. By analysis of an additional 30 primary human RCCs, mutation A441G was found in 4 of 35 tumors and tumor cell lines (11.5%) but not in the respective normal kidney tissues. By enzymatic digestion, mutation A441G proved to be heterozygous, suggesting a dominant active function. This was supported by showing that stable over‐expression of mutated A441G‐Tiam1 induced transformation of NIH3T3 cells, as determined in a colony formation assay, whereas empty vector and wild‐type Tiam1 failed to do so. In conclusion, a distinct Tiam1 mutation (A441G) was identified in several human RCCs. This mutation induced transformation of NIH3T3 cells and, hence, might play a major role in the progression of human RCCs. Further analyses on Tiam1 mutations in human tumors might give new clues to their role in tumor progression. Int. J. Cancer 88:369–376, 2000.

Expression changes in EZH2, but not in BMI-1, SIRT1, DNMT1 or DNMT3B are associated with DNA methylation changes in prostate cancer

The polycomb proteins BMI-1, EZH2, and SIRT1 are characteristic components of the PRC1, PRC2, and PRC4 repressor complexes, respectively, that modify chromatin. Moreover, EZH2 may influence DNA methylation by direct interaction with DNA methyltransferases. EZH2 expression increases during prostate cancer progression, whereas BMI-1 and SIRT1 are not well investigated. Like EZH2 expression, DNA methylation alterations escalate in higher stage prostate cancers, raising the question whether these epigenetic changes are related. Expression of EZH2, BMI-1, SIRT1, and the DNA methyltransferases DNMT1 and DNMT3B measured by qRT-PCR in 47 primary prostate cancers was compared to APC, ASC, GSTP1, RARB2, and RASSF1A hypermethylation and LINE-1 hypomethylation. SIRT1 and DNMT3B were overexpressed in cancerous over benign tissues, whereas BMI-1 was rather downregulated and DNMT1 significantly diminished. Nevertheless, cancers with higher DNMT1 and BMI-1 expression had worse clinical characteristics, as did those with elevated EZH2. In particular, above median DNMT1 expression predicted a worse prognosis. EZH2 and SIRT1 overexpression were well correlated with increased MKI67. Immunohistochemistry confirmed limited EZH2 and heterogeneous DNMT3B overexpression and explained the decrease in BMI-1 by pronounced heterogeneity among tumor cells. EZH2 overexpression, specifically among all factors investigated, was associated with more frequent hypermethylation, in particular of GSTP1 and RARB2, and also with LINE-1 hypomethylation. Our data reveal complex changes in the composition of polycomb repressor complexes in prostate cancer. Heterogeneously expressed BMI-1 and slightly increased EZH2 may characterize less malignant cancers, whereas more aggressive cases express both at higher levels. SIRT1 appears to be generally increased in prostate cancers. Intriguingly, our data suggest a direct influence of increased EZH2 on altered DNA methylation patterns in prostate cancer.

Prognostic relevance of Tiam1 protein expression in prostate carcinomas

The Rac-specific guanine nucleotide exchange factor, Tiam1, plays a major role in oncogenicity, tumour invasion and metastasis but its usefulness as a prognostic marker in human cancer has not been tested yet. In the present study, Tiam1 expression was analysed in benign secretory epithelium, pre-neoplastic high-grade prostatic intraepithelium neoplasia (HG-PIN) and prostate carcinomas of 60 R0-resected radical prostatectomy specimens by semiquantitative immunohistochemistry. Tiam1 proved significantly overexpressed in both HG-PIN (P<0.001) and prostate carcinomas (P<0.001) when compared to benign secretory epithelium. Strong Tiam1 overexpression (i.e. ⩾3.5-fold) in prostate carcinomas relative to the respective benign prostatic epithelium was statistically significantly associated with disease recurrence (P=0.016), the presence of lymph vessel invasion (P=0.031) and high Gleason scores (GS) (i.e. ⩾7) (P=0.044). Univariate analysis showed a statistically significant association of strong Tiam1 overexpression with decreased disease-free survival (DFS) (P=0.03). This prognostic effect of strong Tiam1 overexpression remained significant in multivariate analysis including preoperative prostate-specific antigen levels, pT stage, and GS (relative risk= 3.75, 95% confidence interval=1.06–13.16; P=0.04). Together, our data suggest that strong Tiam1 overexpression relative to the corresponding benign epithelial cells is a new and independent predictor of decreased DFS for patients with prostate cancer.

Slug/SNAI2 regulates cell proliferation and invasiveness of metastatic prostate cancer cell lines

Many metastatic cancers recapitulate the epithelial-to-mesenchymal transition (EMT) resulting in enhanced cell motility and invasiveness. The EMT is regulated by several transcription factors, including the zinc finger protein SNAI2, also named Slug, which appears to exert additional functions during development and cancer progression. We have studied the function of SNAI2 in prostate cancer cells. Quantitative RT-PCR analysis showed strong SNAI2 expression particularly in the PC-3 and PC3-16 prostate carcinoma cell lines. Knockdown of SNAI2 by specific siRNA induced changes in EMT markers and inhibited invasion of both cell lines into a matrigel matrix. SNAI2 siRNA-treated cells did not tolerate detachment from the culture plates, likely at least in part due to downregulation of integrin α6β4. SNAI2 knockdown disturbed the microtubular and actin cytoskeletons, especially severely in PC-3 cells, resulting in grossly enlarged, flattened, and sometimes multinuclear cells. Knockdown also decreased cell proliferation, with a prominent G0/G1 arrest in PC3-16. Together, our data imply that SNAI2 exerts strong effects on the cytoskeleton and adhesion of those prostate cancer cells that express it and is necessary for their proliferation and invasiveness.

Prognostic relevance of increased Rac GTPase expression in prostate carcinomas

Rac proteins of the Rho-like GTPase family, including the ubiquitous Rac1, the hematopoiesis-specific Rac2, and the least-characterized Rac3 play a major role in oncogenic transformation, tumor invasion and metastasis. However, the prognostic relevance of Rac expression in human tumors has not been investigated yet. In the present study, Rac protein expression was analyzed in benign secretory epithelium, high-grade prostatic intraepithelium neoplasia (HG-PIN), and prostate carcinomas of 60 R0-resected radical prostatectomy specimens by semiquantitative immunohistochemistry. Thus, Rac proteins were significantly strongly expressed in HG-PIN (P < 0.001) and prostate carcinomas (P < 0.001) when compared with benign secretory epithelium. Accordingly, all tumor tissues analyzed by isoform-specific real-time PCR (n = 7) exhibited significantly higher RNA expression levels of Rac (i.e. sum of Rac1 and Rac3 expression levels) than the respective benign counterparts (P = 0.018) and this appeared to result mainly from increased expression of the Rac3 isoform as verified by immunoblotting. Univariate analyses showed statistically significant associations of increased Rac protein expression in prostate cancer (P = 0.045), preoperative prostate-specific antigen levels (P = 0.044), pT stage (P = 0.002), and Gleason score (P = 0.001) with decreased disease-free survival (DFS). This prognostic effect of increased protein expression of Rac remained significant even in a multivariate analysis including all these four factors (relative risk = 3.22, 95% confidence interval = 1.04-10.00; P = 0.043). In conclusion, our data suggest that increased Rac protein expression in prostate cancer relative to the corresponding benign secretory epithelium is an independent predictor of decreased DFS and appears to result mainly from increased expression of the Rac3 isoform.

Protein kinase C in human renal cell carcinomas: role in invasion and differential isoenzyme expression

The role of protein kinase C (PKC) in in vitro invasiveness of four different human renal cell carcinoma (RCC) cell lines of the clear cell type was investigated. Different PKC-inhibitors markedly inhibited invasiveness of the highly invasive cell lines, suggesting an invasion-promoting role of PKC in human RCC. Analysis of PKC-isoenzyme expression by protein fractionation and immunoblotting revealed that all cell lines expressed PKC-α, -ɛ, -ζ, -μ and -ι as known from normal kidney tissue. Interestingly, PKC-δ, known to be expressed by normal kidney epithelial cells of the rat, was absent on protein and RNA levels in all RCC cell lines investigated and in normal human kidney epithelial cells. PKC-ɛ expression levels correlated positively with a high proliferation activity, but no obvious correlation between expression levels of distinct PKC-isoenzymes and in vitro invasiveness was observed. However, by immunofluorescence microscopy, membrane localisation of PKC-α and PKC-ɛ reflecting activation of the enzymes, was associated with a highly invasive potential. In conclusion, our results suggest a role for PKC in invasion of human RCCs and might argue in favour of a particular role of PKC-α and PKC-ɛ. Our results further suggest that organ-specific expression patterns of PKC-isoenzymes are not necessarily conserved during evolution.