Protein kinase Ciota is required for Ras transformation and colon carcinogenesis in vivo

Protein kinase C iota (PKCiota) has been implicated in Ras signaling, however, a role for PKCiota in oncogenic Ras-mediated transformation has not been established. Here, we show that PKCiota is a critical downstream effector of oncogenic Ras in the colonic epithelium. Transgenic mice expressing constitutively active PKCiota in the colon are highly susceptible to carcinogen-induced colon carcinogenesis, whereas mice expressing kinase-deficient PKCiota (kdPKCiota) are resistant to both carcinogen- and oncogenic Ras-mediated carcinogenesis. Expression of kdPKCiota in Ras-transformed rat intestinal epithelial cells blocks oncogenic Ras-mediated activation of Rac1, cellular invasion, and anchorage-independent growth. Constitutively active Rac1 (RacV12) restores invasiveness and anchorage-independent growth in Ras-transformed rat intestinal epithelial cells expressing kdPKCiota. Our data demonstrate that PKCiota is required for oncogenic Ras- and carcinogen-mediated colon carcinogenesis in vivo and define a procarcinogenic signaling axis consisting of Ras, PKCiota, and Rac1.     

Protein kinase CbetaII regulates its own expression in rat intestinal epithelial cells and the colonic epithelium in vivo

Protein kinase C betaII (PKCbetaII) is induced early during colon carcinogenesis. Transgenic mice expressing elevated PKCbetaII in the colonic epithelium (transgenic PKCbetaII mice) exhibit hyperproliferation and enhanced colon carcinogenesis. Here we demonstrate that nullizygous PKCbeta (PKCbetaKO) mice are highly resistant to azoxymethane (AOM)-induced preneoplastic lesions, aberrant crypt foci. However, reexpression of PKCbetaII in the colon of PKCbetaKO mice by transgenesis restores susceptibility to AOM-induced colon carcinogenesis. Expression of human PKCbetaII in rat intestinal epithelial (RIE) cells induces expression of endogenous rat PKCbetaII mRNA and protein. Induction of PKCbetaII is dependent upon catalytically active PKCbetaII and does not appear to involve changes in alternative splicing of the PKCbeta gene. Two human PKCbeta promoter constructs are activated by expression of PKCbetaII in RIE cells. Both PKCbeta promoter activity and PKCbetaII mRNA levels are inhibited by the MEK1 and -2 inhibitor U0126, but not the Cox-2 inhibitor celecoxib in RIE/PKCbetaII cells. PKCbeta promoter activity correlates directly with expression of endogenous PKCbetaII mRNA and protein in HT29 and HCT116 human colon cancer cell lines. PKCbeta promoter activity and PKCbetaII mRNA expression in HCT116 cells are inhibited by the selective PKCbeta inhibitor LY317615 and by U0126, demonstrating autoregulation of PKCbetaII expression. Transgenic PKCbetaII mice exhibit specific induction of endogenous PKCbetaII, but not its splice variant PKCbetaI, in the colonic epithelium in vivo. Taken together, our results demonstrate that 1) expression of PKCbetaII in the colonic epithelium is both necessary and sufficient to confer susceptibility to AOM-induced colon carcinogenesis in transgenic mice, 2) PKCbetaII regulates its own expression in RIE and human colon cancer cells in vitro and in the colonic epithelium in vivo, and 3) PKCbetaII autoregulation is mediated through a MEK-dependent signaling pathway in RIE/PKCbetaII and HCT116 colon cancer cells.     

Protein kinase C betaII and TGFbetaRII in omega-3 fatty acid-mediated inhibition of colon carcinogenesis

Increasing evidence demonstrates that protein kinase C betaII (PKCbetaII) promotes colon carcinogenesis. We previously reported that colonic PKCbetaII is induced during colon carcinogenesis in rodents and humans, and that elevated expression of PKCbetaII in the colon of transgenic mice enhances colon carcinogenesis. Here, we demonstrate that PKCbetaII represses transforming growth factor beta receptor type II (TGFbetaRII) expression and reduces sensitivity to TGF-beta-mediated growth inhibition in intestinal epithelial cells. Transgenic PKCbetaII mice exhibit hyperproliferation, enhanced colon carcinogenesis, and marked repression of TGFbetaRII expression. Chemopreventive dietary omega-3 fatty acids inhibit colonic PKCbetaII activity in vivo and block PKCbetaII-mediated hyperproliferation, enhanced carcinogenesis, and repression of TGFbetaRII expression in the colonic epithelium of transgenic PKCbetaII mice. These data indicate that dietary omega-3 fatty acids prevent colon cancer, at least in part, through inhibition of colonic PKCbetaII signaling and restoration of TGF-beta responsiveness.     

Atypical protein kinase Ciota plays a critical role in human lung cancer cell growth and tumorigenicity

Atypical protein kinase C (aPKC) isozymes function in epithelial cell polarity, proliferation, and survival and have been implicated in cellular transformation. However, the role of these enzymes in human cancer is largely unexplored. Here, we report that aPKCiota is highly expressed in human non-small cell lung cancer cell lines, whereas the closely related aPKC isozyme PKCzeta is undetectable in these cells. Disruption of PKCiota signaling reveals that PKCiota is dispensable for adherent growth of non-small cell lung cancer cells but is required for transformed growth in soft agar in vitro and for tumorigenicity in vivo. Molecular dissection of signaling down-stream of PKCiota demonstrates that Rac1 is a critical molecular target for PKCiota-dependent transformation, whereas PKCiota is not necessary for NFkappaB activation in vitro or in vivo. Expression of the PB1 domain of PKCiota (PKCiota-(1-113)) blocks PKCiota-dependent Rac1 activity and inhibits cellular transformation indicating a role for this domain in the transforming activity of PKCiota. Taken together, our data demonstrate that PKCiota is a critical lung cancer gene that activates a Rac1-->Pak-->Mek1,2-->Erk1,2 signaling pathway required for transformed growth. Our data indicate that PKCiota may be an attractive molecular target for mechanism-based therapies for treatment of lung cancer.     

Signals from the Ras, Rac, and Rho GTPases converge on the Pak protein kinase in Rat-1 fibroblasts

Ras plays a key role in regulating cellular proliferation, differentiation, and transformation. Raf is the major effector of Ras in the Ras > Raf > Mek > extracellular signal-activated kinase (ERK) cascade. A second effector is phosphoinositide 3-OH kinase (PI 3-kinase), which, in turn, activates the small G protein Rac. Rac also has multiple effectors, one of which is the serine threonine kinase Pak (p65(Pak)). Here we show that Ras, but not Raf, activates Pak1 in cotransfection assays of Rat-1 cells but not NIH 3T3 cells. We tested agents that activate or block specific components downstream of Ras and demonstrate a Ras > PI 3-kinase > Rac/Cdc42 > Pak signal. Although these studies suggest that the signal from Ras through PI 3-kinase is sufficient to activate Pak, additional studies suggested that other effectors contribute to Pak activation. RasV12S35 and RasV12G37, two effector mutant proteins which fail to activate PI 3-kinase, did not activate Pak when tested alone but activated Pak when they were cotransfected. Similarly, RacV12H40, an effector mutant that does not bind Pak, and Rho both cooperated with Raf to activate Pak. A dominant negative Rho mutant also inhibited Ras activation of Pak. All combinations of Rac/Raf and Ras/Raf and Rho/Raf effector mutants that transform cells cooperatively stimulated ERK. Cooperation was Pak dependent, since all combinations were inhibited by kinase-deficient Pak mutants in both transformation assays and ERK activation assays. These data suggest that other Ras effectors can collaborate with PI 3-kinase and with each other to activate Pak. Furthermore, the strong correlation between Pak activation and cooperative transformation suggests that Pak activation is necessary, although not sufficient, for cooperative transformation of Rat-1 fibroblasts by Ras, Rac, and Rho.     

Activation of H-Ras and Rac1 correlates with epidermal growth factor-induced invasion in Hs578T and MDA-MB-231 breast carcinoma cells

There is considerable experimental evidence that hyperactive Ras proteins promote breast cancer growth and development including invasiveness, despite the low frequency of mutated forms of Ras in breast cancer. We have previously shown that H-Ras, but not N-Ras, induces an invasive phenotype mediated by small GTPase Rac1 in MCF10A human breast epithelial cells. Epidermal growth factor (EGF) plays an important role in aberrant growth and metastasis formation of many tumor types including breast cancer. The present study aims to investigate the correlation between EGF-induced invasiveness and Ras activation in four widely used breast cancer cell lines. Upon EGF stimulation, invasive abilities and H-Ras activation were significantly increased in Hs578T and MDA-MB-231 cell lines, but not in MDA-MB-453 and T47D cell lines. Using small interfering RNA (siRNA) to target H-Ras, we showed a crucial role of H-Ras in the invasive phenotype induced by EGF in Hs578T and MDA-MB-231 cells. Moreover, siRNA-knockdown of Rac1 significantly inhibited the EGF-induced invasiveness in these cells. Taken together, this study characterized human breast cancer cell lines with regard to the relationship between H-Ras activation and the invasive phenotype induced by EGF. Our data demonstrate that the activation of H-Ras and the downstream molecule Rac1 correlates with EGF-induced breast cancer cell invasion, providing important information on the regulation of malignant progression in mammary carcinoma cells.     

p53-mediated activation of the mitochondrial protease HtrA2/Omi prevents cell invasion

Oncogenic Ras induces cell transformation and promotes an invasive phenotype. The tumor suppressor p53 has a suppressive role in Ras-driven invasion. However, its mechanism remains poorly understood. Here we show that p53 induces activation of the mitochondrial protease high-temperature requirement A2 (HtrA2; also known as Omi) and prevents Ras-driven invasion by modulating the actin cytoskeleton. Oncogenic Ras increases accumulation of p53 in the cytoplasm, which promotes the translocation of p38 mitogen-activated protein kinase (MAPK) into mitochondria and induces phosphorylation of HtrA2/Omi. Concurrently, oncogenic Ras also induces mitochondrial fragmentation, irrespective of p53 expression, causing the release of HtrA2/Omi from mitochondria into the cytosol. Phosphorylated HtrA2/Omi therefore cleaves β-actin and decreases the amount of filamentous actin (F-actin) in the cytosol. This ultimately down-regulates p130 Crk-associated substrate (p130Cas)-mediated lamellipodia formation, countering the invasive phenotype initiated by oncogenic Ras. Our novel findings provide insights into the mechanism by which p53 prevents the malignant progression of transformed cells.     

H-Ras-specific upregulation of granulocyte colony-stimulating factor promotes human breast cell invasion via matrix metalloproteinase-2

Ras expression has been suggested to be a marker for tumor aggressiveness of breast cancer. We previously showed that H-Ras, but not N-Ras, induced invasive/migratory phenotypes in MCF10A human breast epithelial cells. The present study aimed to determine the role of granulocyte colony-stimulating factor in H-Ras-induced malignant progression of human breast epithelial cells. Here, we show that G-CSF plays a crucial role in H-Ras-induced MCF10A cell invasion and migration. The siRNA-mediated knockdown of G-CSF significantly reduced H-Ras-induced matrix metalloproteinase (MMP)-2 expression, as well as invasion/migration, suggesting the functional significance of G-CSF in the invasive phenotype of human breast cells. Importantly, the induction of G-CSF expression conferred the invasive/migratory phenotypes to MCF10A cells with up-regulation of MMP-2 and activation of Rac1, MKK3/6, p38 MAPK, Akt, and ERKs. Knockdown of Rac1 by siRNA significantly inhibited MMP-2 upregulation and invasiveness of G-CSF MCF10A cells, demonstrating that G-CSF-induced MMP-2 upregulation and invasive phenotype is mediated by Rac1. Using human breast tissues and sera from breast cancer patients, we further demonstrate that the expression level of G-CSF is strongly correlated with pathologically-diagnosed breast cancer. These data provide a molecular basis for the crucial role of G-CSF in promoting invasiveness of human breast epithelial cells.     

Colon Macrophages Polarized by Commensal Bacteria Cause Colitis and Cancer through the Bystander Effect

Intestinal commensal bacteria have recently been shown to trigger macrophages to produce diffusible clastogens (or chromosome-breaking factors) through a bystander effect (BSE) that mediates DNA damage and induces chromosomal instability in neighboring cells. Colon macrophages appear central to colon carcinogenesis and BSE through the expression of tumor necrosis factor-α (TNF-α) and cyclooxygenase-2 (COX-2). The former induces netrin-1, a regulator of intestinal epithelial cell apoptosis, and the latter generates trans-4-hydroxy-2-nonenal (4-HNE), an endogenous mutagen. To test whether colon macrophages are key effectors for BSE, we depleted these cells in interleukin-10 knockout mice colonized with Enterococcus faecalis using encapsulated liposomal clodronate (ELC), a bisphosphonate that causes macrophage apoptosis. We observed that E. faecalis polarizes colon macrophages to an M1 phenotype. In addition, depleting these cells suppressed COX-2 and TNF-α, blocked the formation of 4-HNE protein adducts, and inhibited up-regulation of netrin-1—all markers for BSE. Finally, treatment with ELC prevented colitis, β-catenin activation, and cancer formation. These results show that selected human commensals can polarize colon macrophages to the M1 phenotype and, when activated, serve as the key effector for bacterial-induced BSE. Our findings suggest that depleting M1-polarized macro-phages is a mechanism for the chemopreventive activity of bisphosphonates and that it represents a new strategy for preventing colon cancer induced by intestinal commensals.     

Nischarin inhibits Rac induced migration and invasion of epithelial cells by affecting signaling cascades involving PAK

Nischarin, a cytosolic protein that binds the alpha5beta1 integrin, plays an important role in fibroblast migration, and in regulation of the actin cytoskeleton. The effect of Nischarin on Rac induced migration and invasion by breast and colon epithelial cell lines has been determined. In these cells, Rac potently induced migration, as well as invasion of matrix; both of these events were strongly inhibited by overexpression of Nischarin. To understand the mechanism of Nischarin's inhibitory role in Rac induced cell migration, several effector domain mutants of Rac1 were employed. Nischarin was able to inhibit migration induced by Rac effector mutants that can activate PAK and JNK, but not migration stimulated by other Rac mutants. Further, Nischarin inhibited PAK induced cell migration, while not affecting migration induced by MEKK1, a Rac effector in the JNK pathway. In addition, Nischarin failed to inhibit migration induced by MEK1, a downstream effector in the Ras-Raf-MEK-Erk signaling cascade. Furthermore, Nischarin does not affect Rac mediated JNK and PI3K activities. However, Rac induced migration and invasion were effectively blocked by pharmacological inhibitors of PI-3 kinase and MEK. These results suggest that several pathways contribute to cell migration, but that Nischarin selectively inhibits Rac driven signaling cascades that affect migration through PAK.     

Leptin promotes invasiveness of kidney and colonic epithelial cells via phosphoinositide 3-kinase-, rho-, and rac-dependent signaling pathways.

Leptin plays a key role regulating food intake, body weight and fat mass. These critical parameters are associated with an increased risk for digestive and mammary gland cancer in the Western population. Here we determined whether leptin contributes to the invasive phenotype of colonic and kidney epithelial cells at various stages of the neoplastic progression. First, leptin potently (EC50 = 10-30 ng/ml) induces invasion of collagen gels by premalignant familial adenomatous colonic cells PC/AA/C1 and nontumorigenic MDCK kidney epithelial cells, their src-transformed counterparts, and the human adenocarcinoma colonic cells LoVo and HCT-8/S11. Leptin and its Ob-Rb receptors were consistently identified by RT-PCR and immunoblotting in these cell lines, as well as in human colonic epithelial crypts, polyps, colonic tumor resections, and adjacent mucosa. Leptin-induced invasion was effectively blocked by pharmacological inhibitors of several downstream signaling pathways involved in cell transformation, namely, JAK2 tyrosine kinase (AG490), phosphoinositide PI3'-kinase (wortmannin and LY294002), mTOR kinase (rapamycin), and protein kinases C (GF109203X, G?6976). Accordingly, leptin induces transient elevation of the PI3'-kinase lipid products in JAK2 immunoprecipitates prepared from parental MDCK cells. The leptin effect on invasion was potentiated by the activated form of the small GTPase RhoA and was abrogated by dominant negative mutants of RhoA, Rac1, and the p110alpha of PI3'-K. Our data indicate that leptin may exert a local and beneficial effect on migration of normal colonic epithelial cells and reparation of the inflamed or wounded digestive mucosa. We also emphasize a new role for leptin, linking the nutritional and body fat status to digestive cancer susceptibility by stimulating the invasive capacity of colonic epithelial cells at early stages of neoplasia. This finding has potential clinical implications for colon cancer progression and management of obesity.     

The survival kinase Mirk/dyrk1B is activated through Rac1-MKK3 signaling

The serine/threonine kinase Mirk/dyrk1B is activated in several solid tumors where it mediates cell survival, but the mechanism by which Mirk is activated in tumors is unknown. We now demonstrate that Mirk is activated as a kinase by signaling from Rac1 to the mitogen-activated protein kinase kinase MKK3. Rac is a Ras superfamily GTPase that, when activated, functions downstream of Ras oncoproteins to promote cell survival, transformation, and membrane ruffling. The constitutively active mutant Rac1QL activated Mirk in several cell types through MKK3, which in turn activated Mirk by phosphorylation. Dominant negative Rac1, dominant negative MKK3, and knockdown of MKK3 by RNA interference inhibited the kinase activity of co-expressed Mirk. E-cadherin ligation in confluent Madin-Darby canine kidney (MDCK) epithelial cells is known to transiently activate Rac1. Mirk was activated by endogenous Rac1 following E-cadherin ligation in confluent MDCK epithelial cells, whereas treatment of confluent MDCK cells with an Rac1 inhibitor decreased Mirk activity. Disruption of cadherin ligation by EGTA or prevention of cadherin ligation by maintenance of cells at subconfluent density blocked activation of Mirk. Engagement of cadherin molecules on subconfluent cells by an E-cadherin/Fc chimeric molecule transiently activated both Rac1 and Mirk with a similar time course. Rac activity is up-regulated in many human tumors and mediates survival signals, which enable tumor cells to evade apoptosis. This study characterizes a new anti-apoptotic signaling pathway that connects Rac1 with a novel downstream effector, Mirk kinase, which has recently been demonstrated to mediate survival in human tumors.     

Raf and RhoA cooperate to transform intestinal epithelial cells and induce growth resistance to transforming growth factor beta

Although unregulated activation of the Ras/Raf/mitogen-activated protein kinase kinase/Erk signaling pathway is believed to be a central mechanism by which many cell types undergo oncogenic transformation, recent studies indicate that activation of Raf kinase by oncogenic Ras is not sufficient to cause tumorigenic transformation in intestinal epithelial cells. Thus, identification of signaling proteins and pathways that interact with Raf to transform intestinal epithelial cells may be critical for understanding aberrant growth control in the intestinal epithelium. Functional interactions between Raf and the small GTPase RhoA were studied in RIE-1 cells overexpressing both activated Raf(22W) and activated RhoA(63L). Double transfectants were morphologically transformed, formed colonies in soft agar, grew in nude mice, overexpressed cyclin D1 and cyclooxygenase-2 (COX-2), and were resistant to growth inhibition by transforming growth factor (TGF) beta. RIE-Raf and RIE-RhoA single transfectants showed none of these characteristics. Expression of a dominant-negative RhoA(N19) construct in RIE-Ras(12V) cells was associated with markedly reduced COX-2 mRNA, COX-2 protein, and prostaglandin E2 levels when compared with RIE-Ras(12V) cells transfected with vector alone. However, no change in transformed morphology, growth in soft agar, cyclin D1 expression, TGFalpha expression, or TGFbeta sensitivity was observed. In summary, coexpression of activated Raf and RhoA induces transformation and TGFbeta resistance in intestinal epithelial cells. Although blockade of RhoA signaling reverses certain well-described characteristics of RIE-Ras cells, it is insufficient to reverse the transformed phenotype and restore TGFbeta sensitivity. Blockade of additional Rho family members or alternate Ras effector pathways may be necessary to fully reverse the Ras phenotype.     

Oncogenic ras-induced down-regulation of pro-apoptotic protease caspase-2 is required for malignant transformation of intestinal epithelial cells

Resistance of carcinoma cells to anoikis, apoptosis that is normally induced by loss of cell-to-extracellular matrix adhesion, is thought to be essential for the ability of these cells to form primary tumors, invade adjacent tissues, and metastasize to distant organs. Current knowledge about the mechanisms by which cancer cells evade anoikis is far from complete. In an effort to understand these mechanisms, we found that ras, a major oncogene, down-regulates protease caspase-2 (which initiates certain steps of the cellular apoptotic program) in malignant human and rat intestinal epithelial cells. This down-regulation could be reversed by inhibition of a protein kinase Mek, a mediator of Ras signaling. We also found that enforced down-regulation of caspase-2 in nonmalignant intestinal epithelial cells by RNA interference protected them from anoikis. Furthermore, the reversal of the effect of Ras on caspase-2 achieved by the expression of exogenous caspase-2 in detached ras-transformed intestinal epithelial cells promoted well established apoptotic events, such as the release of the pro-apoptotic mitochondrial factors cytochrome c and HtrA2/Omi into the cytoplasm of these cells, significantly enhanced their anoikis susceptibility, and blocked their long term growth in the absence of adhesion to the extracellular matrix. Finally, the blockade of the effect of Ras on caspase-2 substantially suppressed growth of tumors formed by the ras-transformed cells in mice. We conclude that ras-induced down-regulation of caspase-2 represents a novel mechanism by which oncogenic Ras protects malignant intestinal epithelial cells from anoikis, promotes their anchorage-independent growth, and allows them to form tumors in vivo.     

H-Ras-specific activation of Rac-MKK3/6-p38 pathway: its critical role in invasion and migration of breast epithelial cells

Human tumors frequently exhibit constitutively activated Ras signaling, which contributes to the malignant phenotype. Mounting evidence suggests unique roles of the Ras family members, H-Ras, N-Ras and K-Ras, in normal and pathological conditions. In an effort to dissect distinct Ras isoform-specific functions in malignant phenotypic changes, we previously established H-Ras- and N-Ras-activated MCF10A human breast epithelial cell lines. Using these, we showed that p38 kinase is a key signaling molecule differentially regulated between H-Ras and N-Ras, leading to H-Ras-specific induction of invasive and migrative phenotypes. The present study is to further investigate H-Ras- and N-Ras-mediated signaling pathways and to unveil how these pathways are integrated for regulation of invasive/migrative phenotypic conversion of human breast epithelial cells. Here we report that the Rac-MAPK kinase (MKK)3/6-p38 pathway is a unique signaling pathway activated by H-Ras, leading to the invasive/migrative phenotype. In contrast, Raf-MEK-ERK and phosphatidylinositol 3-kinase-Akt pathways, which are fundamental to proliferation and differentiation, are activated by both H-Ras and N-Ras. A significant role for p38 in cell invasion is further supported by the observation that p38 activation by MKK6 transfection is sufficient to induce invasive and migrative phenotypes in MCF10A cells. Activation of the MKK6-p38 pathway results in a marked induction of matrix metalloproteinase (MMP)-2, whereas it had little effect on MMP-9, suggesting MMP-2 up-regulation by MKK6-p38 pathway as a key step for H-Ras-induced invasion and migration. We also provide evidence for cross-talk among the Rac, Raf, and phosphatidylinositol 3-kinase pathways critical for regulation of MMP-2 and MMP-9 expression and invasive phenotype. Taken together, the present study elucidated the role of the Rac-MKK3/6-p38 pathway leading to H-Ras-specific induction of malignant progression in breast epithelial cells, providing implications for developing therapeutic strategies for mammary carcinoma to target Ras downstream signaling molecules required for malignant cancer cell behavior but less critical for normal cell functions.     

Epidermal growth factor stimulates Rac activation through Src and phosphatidylinositol 3-kinase to promote colonic epithelial cell migration

Regulated intestinal epithelial cell migration plays a key role in wound healing and maintenance of a healthy gastrointestinal tract. Epidermal growth factor (EGF) stimulates cell migration and wound closure in intestinal epithelial cells through incompletely understood mechanisms. In this study we investigated the role of the small GTPase Rac in EGF-induced cell migration using an in vitro wound-healing assay. In mouse colonic epithelial (MCE) cell lines, EGF-stimulated wound closure was accompanied by a doubling of the number of cells containing lamellipodial extensions at the wound margin, increased Rac membrane translocation in cells at the wound margin, and rapid Rac activation. Either Rac1 small interfering (si)RNA or a Rac1 inhibitor completely blocked EGF-stimulated wound closure. Whereas EGF failed to activate Rac in colon cells from EGF receptor (EGFR) knockout mice, stable expression of wild-type EGFR restored EGF-stimulated Rac activation and migration. Pharmacological inhibition of either phosphatidylinositol 3-kinase (PI3K) or Src family kinases reduced EGF-stimulated Rac activation. Cotreatment of cells with both inhibitors completely blocked EGF-stimulated Rac activation and localization to the leading edge of cells and lamellipodial extension. Our results present a novel mechanism by which the PI3K and Src signaling cascades cooperate to activate Rac and promote intestinal epithelial cell migration downstream of EGFR.     

Identification of novel Ras-cooperating oncogenes in Drosophila melanogaster: a RhoGEF/Rho-family/JNK pathway is a central driver of tumorigenesis

We have shown previously that mutations in the apico-basal cell polarity regulators cooperate with oncogenic Ras (Ras(ACT)) to promote tumorigenesis in Drosophila melanogaster and mammalian cells. To identify novel genes that cooperate with Ras(ACT) in tumorigenesis, we carried out a genome-wide screen for genes that when overexpressed throughout the developing Drosophila eye enhance Ras(ACT)-driven hyperplasia. Ras(ACT)-cooperating genes identified were Rac1 Rho1, RhoGEF2, pbl, rib, and east, which encode cell morphology regulators. In a clonal setting, which reveals genes conferring a competitive advantage over wild-type cells, only Rac1, an activated allele of Rho1 (Rho1(ACT)), RhoGEF2, and pbl cooperated with Ras(ACT), resulting in reduced differentiation and large invasive tumors. Expression of RhoGEF2 or Rac1 with Ras(ACT) upregulated Jun kinase (JNK) activity, and JNK upregulation was essential for cooperation. However, in the whole-tissue system, upregulation of JNK alone was not sufficient for cooperation with Ras(ACT), while in the clonal setting, JNK upregulation was sufficient for Ras(ACT)-mediated tumorigenesis. JNK upregulation was also sufficient to confer invasive growth of Ras(V12)-expressing mammalian MCF10A breast epithelial cells. Consistent with this, HER2(+) human breast cancers (where human epidermal growth factor 2 is overexpressed and Ras signaling upregulated) show a significant correlation with a signature representing JNK pathway activation. Moreover, our genetic analysis in Drosophila revealed that Rho1 and Rac are important for the cooperation of RhoGEF2 or Pbl overexpression and of mutants in polarity regulators, Dlg and aPKC, with Ras(ACT) in the whole-tissue context. Collectively our analysis reveals the importance of the RhoGEF/Rho-family/JNK pathway in cooperative tumorigenesis with Ras(ACT).     

Actopaxin (α-Parvin) Phosphorylation Is Required for Matrix Degradation and Cancer Cell Invasion

Dysregulation of cell adhesion and motility is known to be an important factor in the development of tumor malignancy. Actopaxin (α-parvin) is a paxillin, integrin-linked kinase, and F-actin binding focal adhesion protein with several serine phosphorylation sites in the amino terminus that contribute to the regulation of cell spreading and migration. Here, phosphorylation of actopaxin is shown to contribute to the regulation of matrix degradation and cell invasion. Osteosarcoma cells stably expressing wild type (WT), nonphosphorylatable (Quint), and phosphomimetic (S4D/S8D) actopaxin demonstrate that actopaxin phosphorylation is necessary for efficient Src and matrix metalloproteinase-driven degradation of extracellular matrix. Rac1 was found to be required for actopaxin-induced matrix degradation whereas inhibition of myosin contractility promoted degradation in the phosphomutant-expressing Quint cells, indicating that a balance of Rho GTPase signaling and regulation of cellular tension are important for the process. Furthermore, actopaxin forms a complex with the Rac1/Cdc42 GEF β-PIX and Rac1/Cdc42 effector PAK1, to regulate actopaxin-dependent matrix degradation. Actopaxin phosphorylation is elevated in the invasive breast cancer cell line MDA-MB-231 compared with normal breast epithelial MCF10A cells. Expression of the nonphosphorylatable Quint actopaxin in MDA-MB-231 cells inhibits cell invasion whereas overexpression of WT actopaxin promotes invasion in MCF10A cells. Taken together, this study demonstrates a new role for actopaxin phosphorylation in matrix degradation and cell invasion via regulation of Rho GTPase signaling.