Macrophages are a key component of the innate immune system. In this study, we investigate how focal adhesion kinase (FAK) and the related kinase Pyk2 integrate adhesion signaling and growth factor receptor signaling to regulate diverse macrophage functions. Primary bone marrow macrophages isolated from mice in which FAK is conditionally deleted from cells of the myeloid lineage exhibited elevated protrusive activity, altered adhesion dynamics, impaired chemotaxis, elevated basal Rac1 activity, and a marked inability to form stable lamellipodia necessary for directional locomotion. The contribution of FAK to macrophage function in vitro was substantiated in vivo by the finding that recruitment of monocytes to sites of inflammation was impaired in the absence of FAK. Decreased Pyk2 expression in primary macrophages also resulted in a diminution of invasive capacity. However, the combined loss of FAK and Pyk2 had no greater effect than the loss of either molecule alone, indicating that both kinases function within the same pathway to promote invasion.

Patient bone mineral density (BMD) predicts the likelihood of osteoporotic fracture. While substantial progress has been made toward elucidating the genetic determinants of BMD, our understanding of the factors involved remains incomplete. Here, using a systems genetics approach in the mouse, we predicted that bicaudal C homolog 1 (Bicc1), which encodes an RNA-binding protein, is responsible for a BMD quantitative trait locus (QTL) located on murine chromosome 10. Consistent with this prediction, mice heterozygous for a null allele of Bicc1 had low BMD. We used a coexpression network-based approach to determine how Bicc1 influences BMD. Based on this analysis, we inferred that Bicc1 was involved in osteoblast differentiation and that polycystic kidney disease 2 (Pkd2) was a downstream target of Bicc1. Knock down of Bicc1 and Pkd2 impaired osteoblastogenesis, and Bicc1 deficiency-dependent osteoblast defects were rescued by Pkd2 overexpression. Last, in 2 human BMD genome-wide association (GWAS) meta-analyses, we identified SNPs in BICC1 and PKD2 that were associated with BMD. These results, in both mice and humans, identify Bicc1 as a genetic determinant of osteoblastogenesis and BMD and suggest that it does so by regulating Pkd2 transcript levels. IntroductionOsteoporosis is a disease characterized by low bone mass, skeletal fragility, and increased risk of fracture (1). Of the traits intrinsic to bone that influence its strength, bone mineral density (BMD) is one of the strongest predictors of fractures (2). BMD is also highly heritable, with approximately 70% of its variation being attributable to genetic factors. As a result, developing a comprehensive understanding of the genes and pathways that regulate BMD promises to lead to novel therapies aimed at preventing and treating bone fragility.Genome-wide association studies (GWAS) have significantly expanded the list of known variants and genes that influence BMD (3). A recent meta-analysis of 17 BMD GWAS (Genetic Effects For Osteoporosis [GEFOS] Consortium) involving approximately 83,000 individuals identified 56 robustly significant associations (4). Interestingly, together, the associations explained less than 5% of the total phenotypic variance in BMD, suggesting that bone mass is highly polygenic and that most of the genes influencing BMD remain to be identified.One strategy that can help fill this knowledge gap is the use of gene discovery in the mouse to inform human BMD GWAS. Several recent studies (5-10) have demonstrated the effectiveness of this strategy. To date, dozens of quantitative trait loci (QTLs) (regions of the genome harboring genetic variation influencing a quantitative trait) affecting BMD have been identified in the mouse (11), and recently the pace of identifying QTL genes has rapidly progressed. This is due in part to the development of sys-

Osteoclasts are highly specialized cells that resorb bone and contribute to bone remodeling. Diseases such as osteoporosis and osteolytic bone metastasis occur when osteoclast-mediated bone resorption takes place in the absence of concurrent bone synthesis. Considerable effort has been placed on identifying molecules that regulate the bone resorption activity of osteoclasts. To this end, we investigated unique and overlapping functions of members of the FAK family (FAK and Pyk2) in osteoclast functions. With the use of a conditional knockout mouse model, in which FAK is selectively targeted for deletion in osteoclast precursors (FAK ⌬myeloid ), we found that loss of FAK resulted in reduced bone resorption by osteoclasts in vitro, coincident with impaired signaling through the CSF-1R. However, bone architecture appeared normal in FAK ⌬myeloid mice, suggesting that Pyk2 might functionally compensate for reduced FAK levels in vivo. This was supported by data showing that podosome adhesion structures, which are essential for bone degradation, were significantly more impaired in osteoclasts when FAK and Pyk2 were reduced than when either molecule was depleted individually. We conclude that FAK contributes to cytokine signaling and bone resorption in osteoclasts and partially compensates for the absence of Pyk2 to maintain proper adhesion structures in these cells.

Breast cancer patients treated with chemotherapy often develop resistance, resulting in highly aggressive tumors that are insensitive to drugs. Elucidating the mechanisms by which cancer cells are able to escape drug-induced apoptosis is thus paramount for developing better approaches to treat and cure breast cancer. Adriamycin is an anthracycline that is regularly used for treatment of breast cancer. Upon exposure to adriamycin, sensitive cells undergo cell death. However, as is the case for many chemotherapeutic drugs, cancer cells frequently develop resistance to adriamycin. We have previously reported that MCF-7 breast cancer cells that overexpress the adaptor molecule p130Cas (Cas) are resistant to adriamycin, whereas MCF-7 cells with endogenous levels of Cas are sensitive to the drug (Ta et al., 2006 Cancer Research). This is consistent with reports showing that high expression of Cas correlates with poor relapse-free and overall survival (Van der Flier et al., 2000 JNCI). Our group has shown that Cas overexpression results in significantly less apoptosis in the presence of adriamycin, and that, the kinase activities of c-Src and PI3K are required for this protection from apoptosis. Furthermore, we show that there is a significant increase in AKT activation when Cas-overexpressing cells are treated with adriamycin. Interestingly, while MCF-7 cells expressing endogenous levels of Cas exhibit a G1 arrest upon adriamycin treatment, the more resistant Cas-overexpressing cells effectively transit through G1 and accumulate in S-phase. This suggests that overexpression of Cas may allow damaged cells to bypass the G0/G1 DNA damage checkpoint. Indeed, both MCF-7 cells expressing either endogenous or elevated levels of Cas show similar degrees of DNA damage when treated with adriamycin, as measured by phospho-histone H2AX. We saw a similar progression through the G0/G1 checkpoint in Cas-overexpressing MCF-7 cells under conditions of serum deprivation, suggesting that high Cas expression may have a relatively global effect on G0/G1 cell cycle checkpoints in response to distinct cellular insults. Based on these data, we hypothesize that Cas overexpression enables breast cancer cells to proceed through the G0/G1 cell cycle arrest induced by conditions of stress, such as DNA damage or nutrient deprivation. Future studies will focus on determining mechanisms by which Cas overexpression allows cancer cells to bypass these cell cycle checkpoints. By gaining a better understanding of how cancer cells are able to adapt and continue to proliferate in an unfavorable environment, and develop resistance to chemotherapy, this study will further the development of better strategies for targeting this aggressive cell population. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3177.

The tumor microenvironment is a critical regulator of tumor progression. Results from a broad spectrum of experimental models and analyses of human tumor specimens provide strong evidence that cells of the myeloid lineage have functional attributes that can potentiate the growth, invasion, and metastasis of cancer. Our approach to investigate the role of the microenvironment in tumor progression is centered on the activity of focal adhesion kinase (FAK) in myeloid lineage cells. We have recently shown in macrophages that FAK, a non-receptor tyrosine kinase, regulates adhesion dynamics and migration toward soluble growth factors and chemokines located at sites of tumor growth. Additionally, mice that have FAK conditionally deleted in this lineage display reduced peritoneal infiltration of macrophages following thioglycollate-induced inflammation. These data suggest that FAK may be involved in regulating the migration of myeloid lineage cells to the tumor and the response of macrophages to factors within the tumor microenvironment. We hypothesize that FAK regulates critical aspects of myeloid/macrophage behavior that allow these cells to support tumor progression and metastasis. This hypothesis is being tested in several tumor models. First, using myeloid-specific conditional FAK knockout mice (FAK-/-), we have tested this hypothesis in the context of both a xenograft tumor model using MDA-MB-231 breast cancer cells and a spontaneous breast tumor model driven by mammary-specific expression of the polyoma virus middle T antigen (MMTV-PyVmT). Significantly fewer tumors developed from orthotopic injection of MDA-MB-231 cells in mice lacking FAK in myeloid lineage cells. F4/80 staining of tumor sections from these mice showed reduced infiltration of macrophages into the tumor mass compared to tumors from wild type (WT) mice. Initial data from the spontaneous MMTV-PyVmT tumor model show no significant differences in time to tumor onset and rate of tumor outgrowth between the FAK-/- and WT mice. Analysis of the lung metastatic tumor burden is ongoing. Second, we have conducted preliminary experiments testing the influence of FAK expression in myeloid lineage cells on the adaptive immune response to tumor growth. Following subcutaneous implantation of the syngeneic B16/F1 melanoma cell line, we observed a significant delay in the onset of tumor formation in FAK-/- mice compared to WT mice. However, when a similar study was performed in a severe combined immunodeficient (SCID) background, tumor outgrowth was identical in WT and conditional knockout mice. Based on these results, we are currently investigating mechanisms through which FAK expression in myeloid lineage cells may regulate the response of lymphocytes to the early stages of tumor outgrowth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1358.