RhoG Signals in Parallel with Rac1 and Cdc42

RhoG is a member of the Rho family of small GTPases and shares high sequence identity with Rac1 and Cdc42. Previous studies suggested that RhoG mediates its effects through activation of Rac1 and Cdc42. To further understand the mechanism of RhoG signaling, we studied its potential activation pathways, downstream signaling properties, and functional relationship to Rac1 and Cdc42 in vivo. First, we determined that RhoG was regulated by guanine nucleotide exchange factors that also activate Rac and/or Cdc42. Vav2 (which activates RhoA, Rac1, and Cdc42) and to a lesser degree Dbs (which activates RhoA and Cdc42) activated RhoG in vitro. Thus, RhoG may be activated concurrently with Rac1 and Cdc42. Second, some effectors of Rac/Cdc42 (IQGAP2, MLK-3, PLD1), but not others (e.g. PAKs, POSH, WASP, Par-6, IRSp53), interacted with RhoG in a GTP-dependent manner. Third, consistent with this differential interaction with effectors, activated RhoG stimulated some (JNK and Akt) but not other (SRF and NF-kappaB) downstream signaling targets of activated Rac1 and Cdc42. Finally, transient transduction of a tat-tagged Rac1(17N) dominant-negative fusion protein inhibited the induction of lamellipodia by the Rac-specific activator, Tiam1, but not by activated RhoG. Together, these data argue that RhoG function is mediated by signals independent of Rac1 and Cdc42 activation and instead by direct utilization of a subset of common effectors

Auto-inhibition of the Dbl Family Protein Tim by an N-terminal Helical Motif

Dbl-related oncoproteins are guanine nucleotide exchange factors specific for Rho-family GTPases and typically possess tandem Dbl homology (DH) and pleckstrin homology domains that act in concert to catalyze exchange. Because the ability of many Dbl-family proteins to catalyze exchange is constitutively activated by truncations N-terminal to their DH domains, it has been proposed that the activity of Dbl-family proteins is regulated by auto-inhibition. However, the exact mechanisms of regulation of Dbl-family proteins remain poorly understood. Here we show that the Dbl-family protein, Tim, is auto-inhibited by a short, helical motif immediately N-terminal to its DH domain, which directly occludes the catalytic surface of the DH domain to prevent GTPase activation. Similar to the distantly related Vav isozymes, auto-inhibition of Tim is relieved by truncation, mutation, or phosphorylation of the auto-inhibitory helix. A peptide comprising the helical motif inhibits the exchange activity of Tim in vitro. Furthermore, substitutions within the most highly conserved surface of the DH domain designed to disrupt interactions with the auto-inhibitory helix also activate the exchange process

Transformation of Different Human Breast Epithelial Cell Types Leads to Distinct Tumor Phenotypes

We investigated the influence of normal cell phenotype on the neoplastic phenotype by comparing tumors derived from two different normal human mammary epithelial cell populations, one of which was isolated using a new culture medium. Transformation of these two cell populations with the same set of genetic elements yielded cells that formed tumor xenografts exhibiting major differences in histopathology, tumorigenicity, and metastatic behavior. While one cell type (HMECs) yielded squamous cell carcinomas, the other cell type (BPECs) yielded tumors closely resembling human breast adenocarcinomas. Transformed BPECs gave rise to lung metastases and were up to 10 4-fold more tumorigenic than transformed HMECs, which are nonmetastatic. Hence, the pre-existing differences between BPECs and HMECs strongly influence the phenotypes of their transformed derivatives

microRNAs converge on the transcription factor FOXP2 and promote breast cancer metastasis

Mesenchymal stem/stromal cells (MSCs) are pro- genitor cells shown to participate in breast tumor stroma formation and to promote metastasis. Despite expanding knowledge of their contributions to breast malignancy, the underlying molecular re- sponses of breast cancer cells (BCCs) to MSC influ- ences remain incompletely understood. Here, we show that MSCs cause aberrant expression of micro- RNAs, which, led by microRNA-199a, provide BCCs with enhanced cancer stem cell (CSC) properties. We demonstrate that such MSC-deregulated micro- RNAs constitute a network that converges on and represses the expression of FOXP2, a forkhead transcription factor tightly associated with speech and language development. FOXP2 knockdown in BCCs was sufficient in promoting CSC propaga- tion, tumor initiation, and metastasis. Importantly, elevated microRNA-199a and depressed FOXP2 expression levels are prominent features of malig- nant clinical breast cancer and are associated signif- icantly with poor survival. Our results identify molec- ular determinants of cancer progression of potential utility in the prognosis and therapy of breast cancer