Tracy Vargo-Gogola

Introduction: The Rho GTPase Cdc42 is overexpressed and hyperactivated in breast tumors compared to normal breast tissue. Cdc42 regulates key processes that are critical for mammary gland morphogenesis and become disrupted during the development, progression, and metastasis of breast cancer. However, the contribution of Cdc42 to normal and neoplastic mammary gland development in vivo remains poorly understood. We were therefore interested in investigating the effects of Cdc42 overexpression on mammary gland morphogenesis as a first step toward understanding how its overexpression may contribute to mammary tumorigenesis. Methods: We developed a tetracycline-regulatable Cdc42 overexpression mouse model in which Cdc42 can be inducibly overexpressed in the developing mammary gland. The effects of Cdc42 overexpression during postnatal mammary gland development were investigated using in vivo and in vitro approaches, including morphometric analysis of wholemounted mammary glands, quantification of histological markers, and primary mammary epithelial cell (MEC) functional and biochemical assays. Results: Analysis of Cdc42-overexpressing mammary glands revealed abnormal terminal end bud (TEB) morphologies, characterized by hyperbudding and trifurcation, and increased side branching within the ductal tree. Quantification of markers of proliferation and apoptosis suggested that these phenotypes were not due to increased cell proliferation or survival. Rather, Cdc42 overexpressing MECs were more migratory and contractile and formed dysmorphic, invasive acini in three-dimensional cultures. Cdc42 and RhoA activities, phosphorylated myosin light chain, and MAPK signaling, which contribute to migration and invasion, were markedly elevated in Cdc42 overexpressing MECs. Interestingly, Cdc42 overexpressing mammary glands displayed several features associated with altered epithelial-stromal interactions, which are known to regulate branching morphogenesis. These included increased stromal thickness and collagen deposition, and stromal cells isolated from Cdc42 overexpressing mammary glands exhibited elevated mRNA expression of extracellular matrix proteins and remodeling enzymes. Conclusions: These data suggest that Cdc42 overexpression disrupts mammary gland branching morphogenesis by altering Rho GTPase and MAPK signaling, leading to increased MEC contractility and migration in association with stromal alterations. Our studies provide insight into how aberrant Cdc42 expression may contribute to mammary tumorigenesis.

Rho GTPases mediate stromal-epithelial interactions that are important for mammary epithelial cell (MEC) morphogenesis. Increased extracellular matrix (ECM) deposition and reorganization affect MEC morphogenesis in a Rho GTPase-dependent manner. Although the effects of altered ECM on MEC morphogenesis have been described, how MECs regulate stromal deposition is not well understood. Previously, we showed that p190B RhoGAP overexpression disrupts mammary gland morphogenesis by inducing hyperbranching in association with stromal alterations. We therefore hypothesized that MEC overexpression of p190B regulates paracrine interactions to impact fibroblast activation. Using a combination of in vivo morphometric and immunohistochemical analyses and primary cell culture assays, we found that p190B overexpression in MECs activates fibroblasts leading to increased collagen, fibronectin, and laminin production and elevated expression of the collagen crosslinking enzyme lysyl oxidase. Phosphorylation of the TGF-β effector SMAD2 and expression of the TGF-β target gene αSma were increased in p190B-associated fibroblasts, suggesting that elevated TGF-β signaling promoted fibroblast activation. Mechanical tension and TGF-β cooperate to activate fibroblasts. Interestingly, active TGF-β was elevated in conditioned medium from p190B overexpressing MECs compared to control MECs, and p190B overexpressing MECs exhibited increased contractility in a collagen gel contraction assay. These data suggest that paracrine signaling from the p190B overexpressing MECs may activate TGF-β signaling in adjacent fibroblasts. In support of this, transfer of conditioned medium from p190B overexpressing MECs onto wildtype fibroblasts or co-culture of p190B overexpressing MECs with wildtype fibroblasts increased SMAD2 phosphorylation and mRNA expression of ECM genes in the fibroblasts when compared to fibroblasts treated with control CM or co-cultured with control MECs. The increased ECM gene expression and SMAD2 phosphorylation were blocked by treatment with a TGF-β receptor inhibitor. Taken together, these data suggest that p190B overexpression in the mammary epithelium induces fibroblast activation via elevated TGF-β paracrine signaling.

Rho GTPases are overexpressed and hyperactivated in many cancers, including breast cancer. Rho proteins, as well as their regulators and effectors, have been implicated in mitosis, and their altered expression promotes mitotic defects and aneuploidy. Previously, we demonstrated that p190B Rho GTPase activating protein (RhoGAP) deficiency inhibits ErbB2-induced mammary tumor formation in mice. Here we describe a novel role for p190B as a regulator of mitosis. We found that p190B localized to centrosomes during interphase and mitosis, and that it is differentially phosphorylated during mitosis. Knockdown of p190B expression in MCF-7 and Hela cells increased the incidence of aberrant microtubule-kinetochore attachments at metaphase, lagging chromosomes at anaphase, and micronucleation, all of which are indicative of aneuploidy. Cell cycle analysis of p190B deficient MCF-7 cells revealed a significant increase in apoptotic cells with a concomitant decrease in cells in G1 and S phase, suggesting that p190B deficient cells die at the G1 to S transition. Chemical inhibition of the Rac GTPase during mitosis reduced the incidence of lagging chromosomes in p190B knockdown cells to levels detected in control cells, suggesting that aberrant Rac activity in the absence of p190B promotes chromosome segregation defects. Taken together, these data suggest that p190B regulates chromosome segregation and apoptosis in cancer cells. We propose that disruption of mitosis may be one mechanism by which p190B deficiency inhibits tumorigenesis.

The Rho GTPase Cdc42 is overexpressed and hyperactivated in breast cancer, and several studies have described mechanisms by which it may promote tumor formation and progression. However, little is ...

Introduction: Rho GTPases are overexpressed and hyperactivated in human breast cancers. Deficiency of p190B RhoGAP, a major inhibitor of the Rho GTPases, inhibits mouse mammary tumor virus long terminal repeat (MMTV)-Neu/ErbB2 mammary tumor formation and progression in part through effects within the stromal environment, suggesting that p190B function is pro-tumorigenic. To further investigate the potential pro-tumorigenic actions of p190B, we examined the effects of exogenous p190B expression within the mammary epithelium on MMTV-Neu tumor formation and progression. Methods: Tetracycline (tet)-regulatable p190B transgenic mice were bred to MMTV-Neu mice, and the effects of exogenous p190B expression on tumor latency, multiplicity, growth rates, angiogenesis, and metastasis were examined. The effects of exogenous p190B expression on cell-matrix adhesion and invasion were tested using non-transformed primary mammary epithelial cells (MECs). Rho GTPase activity, oxidative stress as an indicator of reactive oxygen species (ROS) production, and downstream signaling pathways were analyzed. Results: Altered p190B expression resulted in a two-fold increase in tumor multiplicity and a three-fold increase in metastases compared to control mice indicating that exogenous p190B expression in the mammary epithelium promotes MMTV-Neu mammary tumor formation and progression. Interestingly, non-transformed primary MECs expressing exogenous p190B displayed increased adhesion to laminin and type IV collagen and formed invasive structures in a three-dimensional culture assay. Ras related C3 botulinum toxin 1 (Rac1)-GTP levels were elevated in p190B transgenic tumors whereas Ras homologous A (RhoA) and cell division cycle 42 (Cdc42)-GTP levels were not significantly altered. Rac1 activity affects production of ROS, which regulate transformation, metastasis, and oxidative stress. Protein carbonylation, which is indicative of oxidative stress, was elevated 1.75-fold in p190B transgenic tumors as compared to control tumors suggesting that exogenous p190B expression may affect Rac1-dependent ROS production. Conclusions: These studies indicate that paradoxically, p190B RhoGAP, a major inhibitor of the Rho GTPases in vitro, has pro-tumorigenic functions that enhance MMTV-Neu induced mammary tumor formation and metastasis. Furthermore, exogenous p190B expression enhances cell adhesion and invasion, which may facilitate metastasis. Rac1 activity and oxidative stress are elevated in tumors expressing exogenous p190B suggesting that p190B may promote tumorigenesis through a Rac1/ROS dependent mechanism.

Rho GTPase signaling is altered in human breast tumors, and elevated expression and activation of Rho GTPases correlate with tumor progression, metastasis, and poor prognosis. Here we review the evidence that Rho signaling functions as a key regulator of cell cycle, mitosis, apoptosis, and invasion during breast cancer growth and progression and discuss whether these pleiotropic actions enhance or limit the targetability of this network. We propose that depending on the stage and subtype of breast cancer, targeting Rho signaling may have chemopreventative, antitumor, and anti-metastatic efficacy. An understanding of how Rho signaling is perturbed in specific stages and subtypes of breast cancer and how it functions in the context of the complex in vivo environment during the stochastic process of tumor formation and progression are necessary in order to effectively target this signaling network for breast cancer treatment.

Introduction: Rho signaling regulates key cellular processes including proliferation, survival, and migration, and it has been implicated in the development of many types of cancer including breast cancer. P190B Rho GTPase activating protein (RhoGAP) functions as a major inhibitor of the Rho GTPases. P190B is required for mammary gland morphogenesis, and overexpression of p190B in the mammary gland induces hyperplastic lesions. Hence, we hypothesized that p190B may play a pivotal role in mammary tumorigenesis. Methods: To investigate the effects of loss of p190B function on mammary tumor progression, p190B heterozygous mice were crossed with an MMTV-Neu breast cancer model. Effects of p190B deficiency on tumor latency, multiplicity, growth, preneoplastic progression and metastasis were evaluated. To investigate potential differences in tumor angiogenesis between the two groups, immunohistochemistry to detect von Willebrand factor was performed and quantified. To examine gene expression of potential mediators of the angiogenic switch, an angiogenesis PCR array was utilized and results were confirmed using immunohistochemistry. Finally, reciprocal transplantation of tumor fragments was performed to determine the impact of stromal deficiency of p190B on tumor angiogenesis. Results: P190B deficiency reduced tumor penetrance (53% of p190B^+/-Neu mice vs. 100% of p190B^+/+Neu mice formed tumors) and markedly delayed tumor onset by an average of 46 weeks. Tumor multiplicity was also decreased, but an increase in the number of preneoplastic lesions was detected indicating that p190B deficiency inhibited preneoplastic progression. Angiogenesis was decreased in the p190B heterozygous tumors, and expression of a potent angiogenic inhibitor, thrombospondin-1, was elevated in p190B^+/-Neu mammary glands. Transplantation of p190B^+/-Neu tumor fragments into wild-type recipients restored tumor angiogenesis. Strikingly, p190B^+/+Neu tumor fragments were unable to grow when transplanted into p190B^+/-Neu recipients. Conclusions: These data suggest that p190B haploinsufficiency in the epithelium inhibits MMTV-Neu tumor initiation. Furthermore, p190B deficiency in the vasculature is responsible, in part, for the inhibition of MMTV-Neu tumor progression.