Nicolas Wernert

THE MULTIPLE ROLES OF TUMOUR STROMA

Abstract Since the work of Judah Folkman in the 1970s demonstrating the importance of vascularization on tumour growth the many roles played by tumour stroma have been demonstrated. Vascular endothelial growth factor/vascular permeability factor appears to be the main in vivo inducer of both stromal blood vessels and other components of the tumour stroma. Its action is probably mediated through its strong and long-lasting effect on microvascular permeability leading to fibrin extravasation and organisation (”tumours are wounds that do not heal”). During tumour invasion, stromal fibroblasts participate in the degradation of the extracellular matrix (ECM) by secreting matrix degrading proteases as well as their downstream-activators. Stroma derived factors such as scatter factor/hepatocyte growth factor as well as interactions between neoplastic cells and the ECM can play a role in both tumour cell migration and proliferation. The ECM may also act as a reservoir for growth factors. A novel transcription factor encoded by the c-ets 1 proto-oncogene is likely to be involved in the transcriptional regulation of both tumour vascularization and invasion. This contribution summarizes recent developments in the tumour stroma field.

Diagnostic and Prognostic Information in Prostate Cancer with the Help of a Small Set of Hypermethylated Gene Loci

Purpose: Our study was designed to evaluate promoter CpG island hypermethylation in the diagnosis and prognosis of prostate cancer. Experimental Design: Primary prostate cancers from 53 patients, pelvic lymph nodes, noncancerous prostate tissues, and prostate cell lines were analyzed. Real-time methylation-specific PCR was used to identify CpG island hypermethylation at five promising gene loci (i.e., GSTP1, APC, PTGS2, MDR1, and RASSF1a). Results: At three gene loci (GSTP1, APC, and PTGS1) and CpG island, hypermethylation was highly prevalent in prostate cancers (71-91%), and analysis of receiver operator curves showed that hypermethylation at these three gene loci can distinguish between prostate cancer and noncancerous prostatic tissue (i.e., benign hyperplasia) with a sensitivity of 71.1% to 96.2% and a specificity of 92.9% to 100%. Using sensitive SYBR green methylation-specific PCR technology, we observed a respective 28% and 71% hypermethylation rate at the RASSF1a and MDR1 loci in benign prostate hyperplasia, which may represent early nonaggressive carcinogenesis. Methylation characteristics in prostate cancer metastases (i.e., pelvic lymph nodes) were comparable to the respective primary cancer. Statistical analysis showed no correlation between the methylation status of a single gene locus and clinicopathologic variables (e.g., preoperative prostate specific antigen levels, Gleason score, capsular penetration, involvement of seminal vesicle, and age). In contrast, the methylation of two (GSTP1/APC; GSTP1/PTGS2) or three (GSTP1/APC/PTGS2) gene loci correlated with prognostic indicators (i.e., pathologic stage, extraprostatic extension, and Gleason score, but not with prostate specific antigen levels). Conclusions: Our data suggest that the evaluation of DNA hypermethylation at three gene loci (i.e., GSTP1, APC, and PTGS2) is of diagnostic and prognostic value in prostate cancer.

CpG island hypermethylation at multiple gene sites in diagnosis and prognosis of prostate cancer.

OBJECTIVES CpG island hypermethylation causes gene silencing and could be decisive in prostate carcinogenesis and progression. We investigated its role at multiple gene sites during prostate carcinogenesis. METHODS A quantitative, methylation-specific polymerase chain reaction was used to analyze the hypermethylation patterns at nine gene loci (Annexin2, APC, EDNRB, GSTP1, PTGS2, MDR1, RARbeta, Reprimo, and TIG1) in 80 patients with prostate cancer (PCa) and 26 patients with benign prostatic hyperplasia (BPH). RESULTS Hypermethylation was more frequent in PCa than in BPH tissues (EDNRB, 100% versus 88%; TIG1, 96% versus 12%; RARbeta, 95% versus 35%; GSTP1, 93% versus 15%; APC, 80% versus 50%; MDR1, 80% versus 31%; PTGS2, 68% versus 15%; Reprimo, 59% versus 19%; and Annexin2, 4% versus 0%). TIG1 and GSTP1 hypermethylation distinguished between PCa and BPH with a specificity of greater than 85% and sensitivity of greater than 93%. Hypermethylation at a single gene locus did not correlate with any clinicopathologic variables. In contrast, hypermethylation at two genes (eg, APC and TIG1, APC and GSTP1, APC and PTGS2, APC or MDR, GSTP1 or PTGS2) correlated significantly with the pathologic stage and/or Gleason score (P = 0.033 to 0.045). Hypermethylation at APC and Reprimo, as well as DNA hypermethylation at more than five genes, correlated significantly with the rate of prostate-specific antigen recurrence after radical prostatectomy (P = 0.0078 and P = 0.0074, respectively). CONCLUSIONS Our results have confirmed that the hypermethylation patterns are helpful in the diagnosis and prognosis of PCa. Increases in CpG island hypermethylation at multiple gene sites occur during PCa progression and indicate early biochemical recurrence after radical prostatectomy.

The Ets-1 transcription factor is up-regulated together with MMP 1 and MMP 9 in the stroma of pre-invasive breast cancer.

The first steps of stroma generation are of pivotal importance for carcinogenesis because at this stage are initiated both angiogenesis, the prerequisite for continuous tumour growth, and the proliferation of stromal fibroblasts. These developments contribute to the onset of tumour invasion by secreting several matrix‐degrading proteases. Both angiogenesis and the production of proteases are tightly controlled at several levels; of significant importance is transcription. The Ets‐1 transcription factor transactivates several genes encoding matrix‐degrading proteases and is thought to be involved in both tumour vascularization and invasion. This study therefore investigated, by in situ hybridization and immunohistochemistry, the expression of Ets‐1 and of two of its target genes, encoding matrix metalloproteinase (MMP) 1 and MMP 9, in order to demonstrate a topographical in vivo correlation between the expression of these three genes during breast cancer formation. All three genes were first expressed within both endothelial cells and stromal fibroblasts during the onset of stroma generation around intraductal and intralobular in situ carcinomas and they were significantly up‐regulated in the stroma of invasive ductal and lobular cancers. The results of this study further support the suggested in vivo role of Ets‐1 for both angiogenesis and tumour invasion, via matrix‐degrading proteases which are already expressed during the early stages of breast carcinogenesis. Copyright

Frequent epigenetic inactivation of the RASSF1A tumour suppressor gene in testicular tumours and distinct methylation profiles of seminoma and nonseminoma testicular germ cell tumours.

Testicular germ cell tumours (TGCTs) are histologically heterogeneous neoplasms with variable malignant potential. Previously, we demonstrated frequent 3p allele loss in TGCTs, and recently we and others have shown that the 3p21.3 RASSF1A tumour suppressor gene (TSG) is frequently inactivated by promoter hypermethylation in a wide range of cancers including lung, breast, kidney and neuroblastoma. In order to investigate the role of epigenetic events in the pathogenesis of TGCTs, we analysed the promoter methylation status of RASSF1A and nine other genes that may be epigenetically inactivated in cancer (p16INK4A, APC, MGMT, GSTP1, DAPK, CDH1, CDH13, RARβ and FHIT) in 24 primary TGCTs (28 histologically distinct components). RASSF1A methylation was detected in four of 10 (40%) seminomas and 15 of 18 (83%) nonseminoma TGCT (NSTGCT) components (P=0.0346). None of the other nine candidate genes were methylated in seminomas, but MGMT (44%), APC (29%) and FHIT (29%) were frequently methylated in NSTGCTs. Furthermore, in two mixed germ cell tumours, the NSTGCT component for one demonstrated RASSF1A, APC and CDH13 promoter methylation, but the seminoma component was unmethylated for all genes analysed. In the second mixed germ cell tumour, the NSTGCT component was methylated for RASSF1A and MGMT, while the seminoma component was methylated only for RASSF1A. In all, 61% NSTGCT components but no seminoma samples demonstrated promoter methylation at two or more genes (P=0.0016). These findings are consistent with a multistep model for TGCT pathogenesis in which RASSF1A methylation occurs early in tumorigenesis and additional epigenetic events characterize progression from seminoma to NSTGCTs.

The Ets-1 transcription factor is involved in the development and invasion of malignant melanoma

The Ets-1 transcription factor plays a role in tumor vascularization and invasion by regulating expression of matrix-degrading proteases in endothelial cells and fibroblasts in the tumor stroma. During early embryogenesis, Ets-1 is expressed in migrating neural crest cells from which melanocytes arise. In the present study, we analyzed Ets-1 expression in various melanocytic lesions and investigated its functional importance in malignant melanomas. We found that Ets-1 was upregulated both in vivo and in vitro in malignant melanoma, compared to benign melanocytic lesions and to primary melanocytes. Assessment of DNA-binding and transactivation assays documented a strong Ets activity in melanoma cells. Using an antisense strategy, the expression and activity of Ets-1 were reduced in the melanoma cell line Mel Im. This correlated with a diminished expression of several Ets-1 target genes known to be involved in invasion, such as MMP1, MMP3, uPA and integrin β3. In line with these findings, the invasive potential of the melanoma cells measured in a Boyden Chamber model was reduced up to 60% after Ets-1 blockade. This can be attributed to the role of Ets-1 in transcriptional regulation of factors involved in invasion of melanoma cells. We conclude that over-expression of Ets-1 during melanoma development contributes to the malignant phenotype.

Senescence-Associated Oxidative DNA Damage Promotes the Generation of Neoplastic Cells

Studies on human fibroblasts have led to viewing senescence as a barrier against tumorigenesis. Using keratinocytes, we show here that partially transformed and tumorigenic cells systematically and spontaneously emerge from senescent cultures. We show that these emerging cells are generated from senescent cells, which are still competent for replication, by an unusual budding-mitosis mechanism. We further present data implicating reactive oxygen species that accumulate during senescence as a potential mutagenic motor of this post-senescence emergence. We conclude that senescence and its associated oxidative stress could be a tumor-promoting state for epithelial cells, potentially explaining why the incidence of carcinogenesis dramatically increases with advanced age.

Mitochondrial DNA in serum of patients with prostate cancer : a predictor of biochemical recurrence after prostatectomy

To investigate the role of circulating mitochondrial DNA (mtDNA) in patients with localized prostate cancer, as recent reports show that patients with advanced cancer have increased levels of mtDNA.

Stromal nodules in benign prostatic hyperplasia

OBJECTIVE Stromal nodules from benign prostatic hyperplasia (BPH; n = 375 from autopsy, n = 100 from biopsy specimens) were investigated with regard to cytoskeletal components, topography, vascularization, leukocytic infiltrates, and proliferative activity. METHODS The nature of stromal nodules was studied by histopathology and immunohistochemistry, using antibodies against alpha-actin, desmin, myosin, vimentin, BMA-120, factor VIII, CD3, CD4, CD20, CD34, CD45RO, CD68, PCNA, and MiB1. RESULTS The findings lead to an extended classification of stromal nodules in BPH: immature mesenchymal (IM; 8.8%), fibroblastic (FB; 65.2), fibromuscular (FM; 21.6), and smooth muscular (SM; 4.4%). The different types occurred in all age decades in a similar distribution (FB > FM > IM > SM). Topographical studies (modified zonal subdivision of McNeal) revealed stromal nodules predominantly in the transitional zone (n = 286), less in the central zone (n = 78), occasionally in the peripheral zone (n = 11), and predominantly in the periurethral (n = 287) and less in the intermediate (n = 84) and subcapsular (n = 4) regions. The different types of nodules presented a distinct vascular pattern. FB, FM, and SM nodules showed significantly increased diffuse infiltrates of T lymphocytes-mainly T helper cells (mean 73%)-and an increase of B lymphocytes. Proliferative activity in the nodules was not observed. Stromal nodules were not observed in normal nonhyperplastic prostates; they only occurred in combination with hyperplastic nodular glandular proliferates. CONCLUSIONS The findings are suggestive of a maturational sequence of stromal nodules in BPH and of a possible significance of immunocompetent cells in the development of stromal nodules and further suggest that both stroma and epithelium of the prostate respond with nodular hyperplasia to the stimulus, which causes BPH.

Morphological analogies of fetal prostate stroma and stromal nodules in BPH

A “reawakening” of ontogenetic processes in the development of BPH is still in debate. Therefore, morphological analogies of fetal prostate stroma and nodular stromal proliferates in BPH were investigated.