A Mouse Stromal Response to Tumor Invasion Predicts Prostate and Breast Cancer Patient Survival

Primary and metastatic tumor growth induces host tissue responses that are believed to support tumor progression. Understanding the molecular changes within the tumor microenvironment during tumor progression may therefore be relevant not only for discovering potential therapeutic targets, but also for identifying putative molecular signatures that may improve tumor classification and predict clinical outcome. To selectively address stromal gene expression changes during cancer progression, we performed cDNA microarray analysis of laser-microdissected stromal cells derived from prostate intraepithelial neoplasia (PIN) and invasive cancer in a multistage model of prostate carcinogenesis. Human orthologs of genes identified in the stromal reaction to tumor progression in this mouse model were observed to be expressed in several human cancers, and to cluster prostate and breast cancer patients into groups with statistically different clinical outcomes. Univariate Cox analysis showed that overexpression of these genes is associated with shorter survival and recurrence-free periods. Taken together, our observations provide evidence that the expression signature of the stromal response to tumor invasion in a mouse tumor model can be used to probe human cancer, and to provide a powerful prognostic indicator for some of the most frequent human malignancies

Annexin II regulates multivesicular endosome biogenesis in the degradation pathway of animal cells

Proteins of the annexin family are believed to be involved in membrane-related processes, but their precise functions remain unclear. Here, we have made use of several experimental approaches, including pathological conditions, RNA interference and in vitro transport assays, to study the function of annexin II in the endocytic pathway. We find that annexin II is required for the biogenesis of multivesicular transport intermediates destined for late endosomes, by regulating budding from early endosomes—but not the membrane invagination process. Hence, the protein appears to be a necessary component of the machinery controlling endosomal membrane dynamics and multivesicular endosome biogenesis. We also find that annexin II interacts with cholesterol and that its subcellular distribution is modulated by the subcellular distribution of cholesterol, including in cells from patients with the cholesterol-storage disorder Niemann-Pick C. We conclude that annexin II forms cholesterol-containing platforms on early endosomal membranes, and that these platforms regulate the onset of the degradation pathway in animal cells

Histological pattern of cathepsin D expression in human cancers.

<div><p>Representative images of cathepsin D expression in human cancer samples.</p> <p>(A) prostate (B) breast cancer showing cathepsin D expression (brown) by tumor and stromal cells; (C) large-cell lung carcinoma and (D) lung adenocarcinoma showing cathepsin D expression by tumor cells and to a lesser extent by stromal cells; (E, F), cathepsin D is almost exclusively expressed by stromal cells in small-cell lung carcinomas (E) and large-cell neuroendocrine carcinomas (F), whereas tumor cells are almost devoid of cathepin D expression.</p> <p>Nuclei were counterstained with haematoxylin.</p> <p>Magnification 200×.</p></div

Prognostic value of “stroma up” genes for human prostate cancer.

<div><p>(A) Unsupervised hierarchical clustering of prostate cancer patients (columns) obtained using “stroma up” genes (rows).</p> <p>Red indicates high relative levels of gene expression and green represents low relative levels of gene expression.</p> <p>Genes in the cluster are ordered according to decreasing z values (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000032#pone.0000032.s007" target="_blank">Table S4</a>).</p> <p>“Stroma up” genes divide prostate cancer patients in two main clusters (red and blue); (B) Kaplan-Meier survival analysis of the groups of patients defined by “stroma up” genes shows that the two groups of patients differ significantly in the overall survival time (p = 8.05×10⁻⁵; red, poor prognosis group; blue, good prognosis group). Similar analyses performed using “stroma down” genes can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000032#pone.0000032.s003" target="_blank">Figure S3A</a>.</p></div

Univariate cox analysis.

<div><p>Representative histograms of (A) prostate and (B) breast cancer data sets obtained using univariate cox analysis of the correlation between the level of gene expression and survival time.</p> <p>Histograms show that the distribution of the z variable of “stroma up” genes (purple) is significantly higher than that of all genes present in the chip (green); (C) selected genes obtained by cross-list comparison of Tables S4 and S5 found to have strong predictive value for the survival of breast and prostate cancer patients.</p> <p>Only genes having a p value<0.05 in both tables were selected.</p> <p>The <i>z</i> value (and sign) indicate the strength of the correlation between the expression level of a gene and patient survival: the larger the positive value of <i>z</i> the greater the association of the overexpression of the corresponding gene with poor outcome.</p></div

Prognostic value of “stroma up” genes in different human tumors.

<div><p>(A) Unsupervised hierarchical clustering of breast cancer patients (columns) obtained using “stroma up” genes (rows) ordered according to decreasing z values (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000032#pone.0000032.s008" target="_blank">Table S5</a>).</p> <p>“Stroma up” genes divide breast cancer patients in two main clusters (red and blue). </p> <p>Kaplan-Meier survival analysis of the groups of patients defined by “stroma up” genes shows that the two groups of patients differ significantly in the (B) overall survival time (p = 6.97×10⁻⁵), and (C) metastasis-free time (p = 0.0018). Similar analyses performed using “stroma down” genes can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000032#pone.0000032.s003" target="_blank">Figure S3B,C</a>.</p> <p>(D–F) Kaplan-Meier survival analysis of lung (D), gastric (E), and renal cell carcinoma patients (F) shows that groups of patients defined by “stroma up” genes do not differ significantly in the overall survival time (p>0.05). </p> <p>Red, poor prognosis group; blue, good prognosis group.</p></div

Validation of stromal genes identified by microarray analysis.

<div><p>(A) Quantitative real-time RT-PCR confirmed microarray results for 11 transcripts found to be differentially expressed between PIN and invasive cancer stroma.</p> <p>For a better representation, genes induced in the invasive cancer stroma were calibrated on the PIN stroma, those induced in the PIN stroma were calibrated on the invasive-cancer stroma.</p> <p>(B–G) Immunohistochemical validation of cathepsin D expression in invasive cancer stroma; cathepsin D (brown) was highly expressed in stromal cells (arrowheads) associated with invasive cancer, in contrast to tumor cells (T) where only occasional staining was seen (B,E); cells expressing cathepsin D were positive for vimentin (brown), confirming their mesenchymal origin (C); double staining of cathepsin D (brown) and vimentin (blue) highlighted their co-expression by fibroblasts/myofibroblasts (D); anti-SV40T antibody staining (nuclear, brown), (F), and double anti-cathepsin D/anti-SV40T antibody staining (blue/brown, respectively), (G), further confirmed that cathepsin D expression was primarily in stromal cells.</p> <p>Nuclei were counterstained with haematoxylin (B, C, E, F).</p> <p>Magnification 100× (B–D), 200× (E, G).</p> <p>(h) Western blot analysis confirmed increased expression of cathepsin D, B and Z in fibroblasts derived from CR2-TAg prostate cancers (1) compared to those derived from PIN prostates (2).</p> <p>Samples were collected from 24-week (invasive cancer) and 10-week old (PIN) mice, just as for the microarray experiments.</p></div

Stromal cathepsin D expression promotes PNEC cell migration and proliferation.

<div><p>(A) Quantification of PNEC cell migration in 3D matrigel co-culture with cathepsin D-deficient (CTSD^−/−) or wild-type (CTSD^+/+) fibroblasts after 30h of co-culture; (B) PNEC cell proliferation and (C, D) elongation are increased in the presence of conditioned medium (CM) derived from CTSD^+/+ fibroblasts (C), compared to CM derived from CTSD^−/− fibroblasts (D).</p> <p>Experiments were performed three times, each time in quintuplicate.</p> <p>Representative results are shown.</p> <p>*p<0.05, ***p<0.001, Student t test.</p></div

Histological appearance of PIN and invasive CR2-TAg prostate cancer lesions.

<div><p>(A) prostate glands of a 10-week old CR2-TAg mouse showing flat and tufted patterns of PIN (arrowhead and arrow, respectively), and a paucicellular stroma (S); (B) invasive cancer lesion from a 24-week old mouse where PIN acini have been replaced by solid tumor (T) and an abundant cellular, reactive stroma (S) composed primarily of fibroblasts/myofibroblasts as assessed by vimentin/actin smooth muscle staining (data not shown). </p> <p>Tissue sections were stained using anti-SV40 antibody (brown) and counterstained with haematoxylin.</p> <p>Magnification 100×.</p></div