Teresa Garcia

LDL Receptor-Related Protein 5 (LRP5) Affects Bone Accrual and Eye Development

In humans, low peak bone mass is a significant risk factor for osteoporosis. We report that LRP5, encoding the low-density lipoprotein receptor-related protein 5, affects bone mass accrual during growth. Mutations in LRP5 cause the autosomal recessive disorder osteoporosis-pseudoglioma syndrome (OPPG). We find that OPPG carriers have reduced bone mass when compared to age- and gender-matched controls. We demonstrate LRP5 expression by osteoblasts in situ and show that LRP5 can transduce Wnt signaling in vitro via the canonical pathway. We further show that a mutant-secreted form of LRP5 can reduce bone thickness in mouse calvarial explant cultures. These data indicate that Wnt-mediated signaling via LRP5 affects bone accrual during growth and is important for the establishment of peak bone mass.

A Six-Gene Signature Predicting Breast Cancer Lung Metastasis

The lungs are a frequent target of metastatic breast cancer cells, but the underlying molecular mechanisms are unclear. All existing data were obtained either using statistical association between gene expression measurements found in primary tumors and clinical outcome, or using experimentally derived signatures from mouse tumor models. Here, we describe a distinct approach that consists of using tissue surgically resected from lung metastatic lesions and comparing their gene expression profiles with those from nonpulmonary sites, all coming from breast cancer patients. We show that the gene expression profiles of organ-specific metastatic lesions can be used to predict lung metastasis in breast cancer. We identified a set of 21 lung metastasis-associated genes. Using a cohort of 72 lymph node-negative breast cancer patients, we developed a 6-gene prognostic classifier that discriminated breast primary cancers with a significantly higher risk of lung metastasis. We then validated the predictive ability of the 6-gene signature in 3 independent cohorts of breast cancers consisting of a total of 721 patients. Finally, we show that the signature improves risk stratification independently of known standard clinical variables and a previously established lung metastasis signature based on an experimental breast cancer metastasis model.

Identification of genes regulated during osteoblastic differentiation by genome-wide expression analysis of mouse calvaria primary osteoblasts in vitro

Although several independent studies of gene expression patterns during osteoblast differentiation in cultures from calvaria and other in vitro models have been reported, only a small portion of the mRNAs expressed in osteoblasts have been characterized. We have previously analyzed the behavior of several known markers in osteoblasts, using Affymetrix GeneChip murine probe arrays (27,000 genes). In the present study we report larger groups of transcripts displaying significant expression modulation during the culture of osteoblasts isolated from mice calvaria. The expression profiles of 601 such regulated genes, classified in distinct functional families, are presented and analyzed here. Although some of these genes have previously been shown to play important roles in bone biology, the large majority of them have never been demonstrated to be regulated during osteoblast differentiation. Despite the fact that the precise involvement of these genes in osteoblast differentiation and function needs to be evaluated, the data presented herein will aid in the identification of genes that play a significant role in osteoblasts. This will provide a better understanding of the regulation of osteoblast differentiation and maturation.

Murine frizzled-1 behaves as an antagonist of the canonical Wnt/β-catenin signaling

Activation of the Wnt signaling cascade provides key signals during development and in disease. Wnt signals are transduced by seven-transmembrane Frizzleds (Fzs) and the single transmembrane low density lipoprotein receptor-related proteins 5 or 6. In the course of the analysis of genes regulated by bone morphogenetic protein 2 in mesenchymal cells we found a significant induction of murine Frizzled-1 (mFz1) gene expression. Unexpectedly overexpression of mFz1 dramatically repressed the induction of alkaline phosphatase mediated by either bone morphogenetic protein 2 or Wnt3a in these cells. Moreover mFz1 overexpression significantly repressed both β-catenin translocation into the nucleus and T cell factor signaling mediated by Wnt3a. Importantly microinjection of mFz1 transcript in Xenopus embryo inhibited the ability of Wnt1 to induce the expression of the Wnt/β-catenin target gene Siamois in animal cap assay and secondary axis formation in whole embryo. By using chimeric constructs in which N- and C-terminal segments of mFz1 were replaced by the corresponding parts of Xfz3 we demonstrated that the antagonistic activity resides in the cysteine-rich domain of the N-terminal part. The antagonist activity of mFz1 could be prevented by overexpression of Gαq-(305-359), which specifically uncouples Gq-coupled receptors, suggesting that Gαq signaling contributes to the inhibition of Wnt/β-catenin pathway by mFz1. This is the first time that a Frizzled receptor has been reported to antagonize Wnt/β-catenin.

Increased expression of a set of genes enriched in oxygen binding function discloses a predisposition of breast cancer bone metastases to generate metastasis spread in multiple organs

Bone is the preferential site of distant metastasis in breast carcinoma (BrCa). Patients with metastasis restricted to bone (BO) usually show a longer overall survival compared to patients who rapidly develop multiple metastases also involving liver and lung. Hence, molecular predisposition to generate bone and visceral metastases (BV) represents a clear indication of poor clinical outcome. We performed microarray analysis with two different chip platforms, Affymetrix and Agilent, on bone metastasis samples from BO and BV patients. The unsupervised hierarchical clustering of the resulting transcriptomes correlated with the clinical progression, segregating the BO from the BV profiles. Matching the twofold significantly regulated genes from Affymetrix and Agilent chips resulted in a 15‐gene signature with 13 upregulated and two downregulated genes in BV versus BO bone metastasis samples. In order to validate the resulting signature, we isolated different MDA‐MB‐231 clonal subpopulations that metastasize only in the bone (MDA‐BO) or in bone and visceral tissues (MDA‐BV). Six of the signature genes were also significantly upregulated in MDA‐BV compared to MDA‐BO clones. A group of upregulated genes, including Hemoglobin B (HBB), were involved in oxygen metabolism, and in vitro functional analysis of HBB revealed that its expression in the MDA subpopulations was associated with a reduced production of hydrogen peroxide. Expression of HBB was detected in primary BrCa tissue but not in normal breast epithelial cells. Metastatic lymph nodes were frequently more positive for HBB compared to the corresponding primary tumors, whereas BO metastases had a lower expression than BV metastases, suggesting a positive correlation between HBB and ability of bone metastasis to rapidly spread to other organs. We propose that HBB, along with other genes involved in oxygen metabolism, confers a more aggressive metastatic phenotype in BrCa cells disseminated to bone.