Few mutations link well defined behaviors with individual neurons and the activity of specific genes. In Drosophila, recent evidence indicates the presence of a doublesex-independent pathway controlling sexual behavior and neuronal differentiation. We have identified a gene, dissatisfaction (dsf), that affects sex-specific courtship behaviors and neural differentiation in both sexes without an associated general behavioral debilitation. Male and female mutant animals exhibit abnormalities in courtship behaviors, suggesting a requirement for dsf in the brain. Virgin dsf females resist males during courtship and copulation and fail to lay mature eggs. dsf males actively court and attempt copulation with both mature males and females but are slow to copulate because of maladroit abdominal curling. Structural abnormalities in specific neurons indicate a role for dsf in the differentiation of sex-specific abdominal neurons. The egglaying defect in females correlates with the absence of motor neuronal innervation on uterine muscles, and the reduced abdominal curling in males correlates with alteration in motor neuronal innervation of male ventral abdominal muscles. Epistasis experiments show that dsf acts in a tra-dependent and dsx-independent manner, placing dsf in the dsxindependent portion of the sex determination cascade.
Transforming growth factor  (TGF-) receptor (TR) signaling contributes to normal development as well as tumorigenesis. Here we report that RIN1, a RAB5 guanine nucleotide exchange factor (GEF) and down regulator of receptor tyrosine kinases (RTKs), promotes TR signaling through enhanced endocytosis. TR activation induces SNAI1 (Snail), a transcription repressor that reduces RIN1 expression, providing a negative feedback mechanism to control TR trafficking and downstream signaling. Persistent RAS signaling disrupts this equilibrium by stabilizing SNAI1 protein, resulting in strong silencing of RIN1 and stabilization of RTKs. TGF--induced RIN1 silencing in breast cancer cells prolonged sensitivity to hepatocyte growth factor, a ligand for the MET-type RTK, and enhanced growth factor-directed cell motility. We conclude that in some tumor cells TR and RAS signals are integrated through the silencing of RIN1, leading to a reduction in RAB5-mediated endocytosis. These findings shed new light on the basis for distinct interpretations of TGF- signaling by normal versus transformed cells.Cell surface receptor internalization controls the intensity and duration of signaling and, as a consequence, regulates phenotypes such as differentiation, mitosis, and migration (36, 44). The clathrin-dependent endocytosis of receptor tyrosine kinases (RTKs), for example, typically results in down regulation and signal termination. The GTPase RAB5 is a key regulator of early endosome formation and trafficking and hence a control point for receptor function. This small GTPase is itself activated by the guanine nucleotide exchange factor (GEF) RIN1 (50) and other GEFs (9) and inhibited by GTPaseactivating proteins (24,39).Transforming growth factor  (TGF-), signaling through a receptor serine/threonine kinase complex (TRI/TRII or TR), has antiproliferative effects on normal epithelial cells (48). Some late-stage tumor cells escape this cytostatic effect and instead respond to TGF- with increased proliferation and enhanced motility that promote metastatic spread (16). Activated TR phosphorylates associated SMAD2 and SMAD3 proteins that then accumulate in the nucleus, inducing expression of SNAI1 (Snail) (42), a promoter of cell motility in development and tumor progression (3), and other genes. This signaling pathway is enhanced by the recruitment of effector proteins during clathrin-mediated endocytosis (18,27,38,43). An alternate, clathrin-independent, TR internalization pathway targets TR to a degradation shunt (46). We describe a dynamic role for RIN1 in TR endocytosis and regulation of downstream signal intensity. In addition, we provide evidence that TR and RTK signals integrate through down regulation of RIN1, contributing to the profound difference in TGF- response between normal and transformed cells. MATERIALS AND METHODSCell culture, transfections, and transductions. MCF10A, MDA-MB-231, HeLa, and HepG2 cell lines were cultured under standard conditions. Transfections were performed using Polyfect (Qiagen) (M...
Breast cancer progression is driven by altered gene expression. We show that the RIN1 gene, which encodes a RAS effector regulating epithelial cell properties, is silenced in breast tumor cell lines compared with cultured human mammary epithelial cells. We also report that RIN1 is often reduced in human breast tumor cells compared with morphologically normal breast glandular cells. At least two silencing mechanisms seem to be involved. Overexpression of the transcription repressor SNAI1 (Snail) was observed in ZR75-1 cells, and SNAI1 knockdown restored RIN1 expression. In addition, DNA methylation within the RIN1 promoter and the first exon in KPL-1 cells suggested that epigenetic modifications may contribute to silencing, and demethylation was shown to restore RIN1 expression. Reexpression of RIN1 was shown to inhibit anchorage-independent growth in soft agar. In addition, RIN1 expression inhibited both the initiation and progression of tumorigenesis for two breast tumor cell lines in a mouse model, consistent with a tumor suppressor function. We also show that RIN1 acts as a negative regulator of tumor cell invasive growth and that this requires the ABL kinase-signaling function of RIN1, suggesting a mechanism through which RIN1 silencing may contribute to breast cancer progression. [Cancer Res 2007;67(24):11510-6]
<div>Abstract<p>Breast cancer progression is driven by altered gene expression. We show that the <i>RIN1</i> gene, which encodes a RAS effector regulating epithelial cell properties, is silenced in breast tumor cell lines compared with cultured human mammary epithelial cells. We also report that RIN1 is often reduced in human breast tumor cells compared with morphologically normal breast glandular cells. At least two silencing mechanisms seem to be involved. Overexpression of the transcription repressor <i>SNAI1</i> (Snail) was observed in ZR75-1 cells, and <i>SNAI1</i> knockdown restored <i>RIN1</i> expression. In addition, DNA methylation within the <i>RIN1</i> promoter and the first exon in KPL-1 cells suggested that epigenetic modifications may contribute to silencing, and demethylation was shown to restore RIN1 expression. Reexpression of <i>RIN1</i> was shown to inhibit anchorage-independent growth in soft agar. In addition, RIN1 expression inhibited both the initiation and progression of tumorigenesis for two breast tumor cell lines in a mouse model, consistent with a tumor suppressor function. We also show that RIN1 acts as a negative regulator of tumor cell invasive growth and that this requires the ABL kinase–signaling function of RIN1, suggesting a mechanism through which <i>RIN1</i> silencing may contribute to breast cancer progression. [Cancer Res 2007;67(24):11510–6]</p></div>
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