Combined mutation of Vhl and Trp53 causes renal cysts and tumours in mice

The combinations of genetic alterations that cooperate with von Hippel-Lindau (VHL) mutation to cause clear cell renal cell carcinoma (ccRCC) remain poorly understood. We show that the TP53 tumour suppressor gene is mutated in approximately 9% of human ccRCCs. Combined deletion of Vhl and Trp53 in primary mouse embryo fibroblasts causes proliferative dysregulation and high rates of aneuploidy. Deletion of these genes in the epithelium of the kidney induces the formation of simple cysts, atypical cysts and neoplasms, and deletion in the epithelia of the genital urinary tract leads to dysplasia and tumour formation. Kidney cysts display a reduced frequency of primary cilia and atypical cysts and neoplasms exhibit a pro-proliferative signature including activation of mTORC1 and high expression of Myc, mimicking several cellular and molecular alterations seen in human ccRCC and its precursor lesions. As the majority of ccRCC is associated with functional inactivation of VHL, our findings suggest that for a subset of ccRCC, loss of p53 function represents a critical event in tumour development

IGF-I induced genes in stromal fibroblasts predict the clinical outcome of breast and lung cancer patients

<p>Abstract</p> <p>Background</p> <p>Insulin-like growth factor-1 (IGF-I) signalling is important for cancer initiation and progression. Given the emerging evidence for the role of the stroma in these processes, we aimed to characterize the effects of IGF-I on cancer cells and stromal cells separately.</p> <p>Methods</p> <p>We used an <it>ex vivo </it>culture model and measured gene expression changes after IGF-I stimulation with cDNA microarrays. <it>In vitro </it>data were correlated with <it>in vivo </it>findings by comparing the results with published expression datasets on human cancer biopsies.</p> <p>Results</p> <p>Upon stimulation with IGF-I, breast cancer cells and stromal fibroblasts show some common and other distinct response patterns. Among the up-regulated genes in the stromal fibroblasts we observed a significant enrichment in proliferation associated genes. The expression of the IGF-I induced genes was coherent and it provided a basis for the segregation of the patients into two groups. Patients with tumours with highly expressed IGF-I induced genes had a significantly lower survival rate than patients whose tumours showed lower levels of IGF-I induced gene expression (<it>P </it>= 0.029 - Norway/Stanford and <it>P </it>= 7.96e-09 - NKI dataset). Furthermore, based on an IGF-I induced gene expression signature derived from primary lung fibroblasts, a separation of prognostically different lung cancers was possible (<it>P </it>= 0.007 - Bhattacharjee and <it>P </it>= 0.008 - Garber dataset).</p> <p>Conclusion</p> <p>Expression patterns of genes induced by IGF-I in primary breast and lung fibroblasts accurately predict outcomes in breast and lung cancer patients. Furthermore, these IGF-I induced gene signatures derived from stromal fibroblasts might be promising predictors for the response to IGF-I targeted therapies.</p> <p>See the related commentary by Werner and Bruchim: <url>http://www.biomedcentral.com/1741-7015/8/2</url></p

Tumor-stroma derived gene expression patterns as prognosticators in breast cancer

The stroma, which embeds epithelial cells, plays a major role in the shaping and physiological regulation of animal organs such as the breast. Aberrant functioning of the stroma was found to support tumorigenesis and cancer progression. Among many crucial aspects of breast cancer biology two issues focused our attention; (I) the impact of mutual interactions between the cancer cells and the stroma, specifically the endothelial cells and (II) the impact of insulin like growth factor one (IGF-I) on the gene expression profiles of stromal fibroblasts, as an example of the influence of a growth factor on the stroma. To address tumor-endothelial interaction we took advantage of an in vitro system mimicking the interaction of tumor and endothelial cells and explored gene expression changes using DNA microarrays. Our results suggest the interaction of endothelial cells and tumor cells that express the CD44+/CD24- signature indicative of stem cell-like cells, which are thought to have a low proliferative potential, might explain the unexpected and paradoxical association of the CD44+/CD24- signature with highly proliferative tumors that have an unfavorable prognosis. Furthermore, the gene expression signature induced in this system is of prognostic value in early stage and metastatic breast cancer. Since malignant epithelial cells and tumor-associated stromal cells are under the influence of hormones and growth factors, we examined the effects of IGF-I on cancer cells and primary fibroblasts in parallel to assess concordant and discordant gene expression changes. Our results show that primary breast fibroblasts, breast carcinoma associated fibroblasts and primary lung fibroblasts respond to IGF-I stimulation with increased expression of genes related with proliferation, which is phenotypically followed by an increased growth rate. Comparing in vitro gene expression data with available in vivo data, we have shown that the evoked gene expression signatures are able to stratify patients into groups with significantly different outcome. We propose that this prognostic gene expression signature might also serve as a predictor for the effectiveness of an anti-IGF-I therapy, a new therapeutic strategy that is currently in phase III development. Concluding, the data presented in this thesis underline the importance of the stroma, specifically the interaction between tumor and endothelial cells and the response of the stroma to stimulation with growth factors such as IGF-I. The gene expression changes in response to these interactions and stimulations carry prognostic information and might potentially be useful in clinical decision making

Vhl deletion in renal epithelia causes HIF-1α-dependent, HIF- 2α-independent angiogenesis and constitutive diuresis

One of the earliest requirements for the formation of a solid tumor is the establishment of an adequate blood supply. Clear cell renal cell carcinomas (ccRCC) are highly vascularized tumors in which the earliest genetic event is most commonly the biallelic inactivation of the VHL tumor suppressor gene, leading to constitutive activation of the HIF-1α and HIF-2α transcription factors, which are known angiogenic factors. However it remains unclear whether either or both HIF-1α or HIF-2α stabilization in normal renal epithelial cells are necessary or sufficient for alterations in blood vessel formation. We show that renal epithelium-specific deletion of Vhl in mice causes increased medullary vascularization and that this phenotype is completely rescued by Hif1a co-deletion, but not by co-deletion of Hif2a. A physiological consequence of changes in the blood vessels of the vasa recta in Vhl-deficient mice is a diabetes insipidus phenotype of excretion of large amounts of highly diluted urine. This constitutive diuresis is fully compensated by increased water consumption and mice do not show any signs of dehydration, renal failure or salt wasting and blood electrolyte levels remain unchanged. Co-deletion of Hif1a, but not Hif2a, with Vhl, fully restored kidney morphology and function, correlating with the rescue of the vasculature. We hypothesize that the increased medullary vasculature alters salt uptake from the renal interstitium, resulting in a disruption of the osmotic gradient and impaired urinary concentration. Taken together, our study characterizes a new mouse model for a form of diabetes insipidus and non-obstructive hydronephrosis and provides new insights into the physiological and pathophysiological effects of HIF-1α stabilization on the vasculature in the kidney

Global Gene Expression Analysis of the Interaction between Cancer Cells and Osteoblasts to Predict Bone Metastasis in Breast Cancer

Background: Bone metastasis is a main cause of morbidity in breast cancer. Since breast cancer is a heterogeneous disease, the interactions of cancer cells with the skeletal host cells might also be diverse. We hypothesized that gene expression signatures induced by heterotypic interaction of breast cancer cells and osteoblasts might be of clinical relevance. Methodology/Principal Findings: We established an ex vivo co-culture model using benign breast epithelial cells or a panel of 5 malignant breast epithelial cells in combination with primary human osteoblasts and determined associated gene expression changes with HEEBO microarrays. Pretreatment gene expression profiles of 295 early stage breast cancers published from the Netherlands Cancer Institute with a median follow up of 12.6 years allowed evaluating in vitro effects in the in vivo situation.The effects of the interaction between osteoblasts and breast cancer cell lines of different origin were very heterogeneous. Hs578T cells started to proliferate in co-culture with osteoblasts, SKBR-3 induced a TGF-b response and MDA-MB231 cells showed two distinct sets of up-regulated genes: A set of interferon response genes associated with an upregulation of STAT1 was in vivo remarkably coherent providing a basis for segregation of tumors into two groups. In a univariate analysis, early stage tumors with high expression levels (n = 136) of this gene set had a significantly lower overall survival rate (p = 0.005) (63 % at 10 years) than tumors with low expression levels (n = 159) (overall survival: 77 % at 10 years). The second gene set was associated with IL-6 and did not significantly change the overall survival rate (p = 0.165), but wa

Tumor-endothelial interaction links the CD44(+)/CD24(-) phenotype with poor prognosis in early-stage breast cancer

MATERIALS AND METHODS: The genomic effects of tumor-endothelial interactions in cancer are not yet well characterized. To study this interaction in breast cancer, we set up an ex vivo coculture model with human benign and malignant breast epithelial cells with endothelial cells to determine the associated gene expression changes using DNA microarrays. RESULTS: The most prominent response to coculture was the induction of the M-phase cell cycle genes in a subset of breast cancer cocultures that were absent in cocultures with normal breast epithelial cells. In monoculture, tumor cells that contained the stem cell-like CD44(+)/CD24(-) signature had a lower expression of the M-phase cell cycle genes than the CD44(-)/CD24(+) cells, and in the CD44(+)/CD24(-) cocultures, these genes were induced. Pretreatment gene expression profiles of early-stage breast cancers allowed evaluating in vitro effects in vivo. The expression of the gene set derived from the coculture provided a basis for the segregation of the tumors into two groups. In a univariate analysis, early-stage tumors with high expression levels (n = 137) of the M-phase cell cycle genes had a significantly lower metastasis-free survival rate (P = 1.8e - 5, 50% at 10 years) and overall survival rate (P = 5e - 9, 52% at 10 years) than tumors with low expression (n = 158; metastasis-free survival, 73%; overall survival, 84%). CONCLUSIONS: Our results suggest that the interaction of endothelial cells with tumor cells that express the CD44(+)/CD24(-) signature, which indicates a low proliferative potential, might explain the unexpected and paradoxical association of the CD44(+)/CD24(-) signature with highly proliferative tumors that have an unfavorable prognosis

Effects of a heterotypic interaction between normal human osteoblasts and breast cancer cells.

<p>(<b>A</b>) Biologically independent replicates of NHOst, Hs578T, and the mixed co-culture of NHOst and Hs578T were kept for 48 hours at low serum conditions and characterized by DNA microarray profiling. We performed hierarchical clustering of 1923 elements that display a greater than 3-fold variance in expression in more than 2 different experimental samples. Genes are represented in rows and experiments in columns. Unsupervised hierarchical clustering of the experiments grouped the biological replicates together. The vertical black bar marks a cluster of genes that were higher expressed in all co-cultures compared to both monocultures, which indicated that they were induced by the heterotypic interaction. Further analysis of these genes revealed that they were specific for proliferation and mitosis. (<b>B</b>) The proliferation rate of NHOst and Hs578T cell monocultures and of their 1∶1 co-culture was determined by measuring increases in cell number by direct cell counting. Quadruplicates of pre-starved cells were plated at a density of 8500 cells/cm² and after 48 hours the cell number was determined using a cell counter All figures represent averages from four replicates, and error bars denote standard deviation. The increase in cell number of the co-culture is significantly higher than the increase in both of the monocultures (p = 0.0084, un-paired, two-tailed <i>t</i>-test). (<b>C</b>) Hs578T cells and NHOst cells were incubated for 24 hours with conditioned media (CM) from NHOst cells or Hs578T cells, respectively, and compared to a negative control of the same cells incubated with autologous medium. All experiments were performed in triplicates. After 24 hours cell numbers were measured by the cell counting with FACS. (<b>D</b>) The expression values of the genes in the “tumor-osteoblast cell-induced M phase/cell cycle” gene signature were extracted from a published expression study of 295 early stage breast cancers from the Netherlands Cancer Institute. Genes and samples were organized by hierarchical clustering. The tumors were segregated into two groups that were defined by high or low expression levels of the 36 genes matching the proliferation gene cluster. The histogram below the heat map represents the differences in the sums of log2 ratios among groups. Based on the distributions, the sums of the log2 ratios for the “proliferation” transcripts were over-expressed in the majority of the cases in the right branch of the cluster compared to the left branch of the cluster (32/113 versus 105/45 scores below/above reference zero value, respectively). (<b>E+F</b>) Correlation of the “proliferation” gene signature with distant metastasis-free survival (DMFS) (E) and overall survival (OS) (F). Kaplan-Meier curves for the clinical outcomes of the indicated tumors that exhibited high (red curve) and low (black curve) expression of the “proliferation” signature are shown.</p

“Interferon response genes regulated by STAT1 signature” and “IL-6 gene signature” in co-culture.

<p>(<b>A</b>) Biologically independent replicates of the monocultured normal human osteoblasts (NHOst), the breast cancer cell line MDA-MB-231, and the mixed co-culture of NHOst and MDA-MB-231 cells were incubated for 48 hours under low serum conditions and characterized by DNA microarray hybridization. The figure shows the heat map of the hierarchical clustering of a total of 1461 elements that display a greater than 1.5-fold variance in expression of at least 2 different experimental samples. The co-culture of NHOst and MDA-MB231 induced two prominent sets of genes: An “interferon–response genes regulated by STAT1” signature and an “IL-6 gene signature” (zoomed image). (<b>B</b>) Real time PCR confirms a significantly higher expression of IL-6 mRNA in the co-culture than in either of the two monocultures (p = 4e-7; un-paired, two-tailed <i>t</i>-test). (<b>C</b>) As implied by the higher expression levels of IL-6 mRNA, the IL-6 concentration in the co-culture supernatants, as determined by ELISA, were significantly higher than the average concentration of the two monocultures. (p = 0.0046; un-paired, two-tailed <i>t</i>-test). (<b>D</b>) Also IL-6 was significantly more highly induced in NHOst cells stimulated with conditioned medium from MDA-MB-231 cells than in MDA-MB-231 cells stimulated with conditioned medium from NHOst (p = 0.045; un-paired, two-tailed <i>t</i>-test).</p