3Y1 rat fibroblasts overexpressing the epidermal growth factor (EGF) receptor (EGFR cells) become transformed when treated with EGF.A common response to oncogenic and mitogenic stimuli is elevated phospholipase D (PLD) activity. RalA, a small GTPase that functions as a downstream effector molecule of Ras, exists in a complex with PLD1. In the EGFR cells, EGF induced a Ras-dependent activation of RalA. The activation of PLD by EGF in these cells was dependent upon both Ras and RalA. In contrast, EGF-induced activation of Erk1, Erk2, and Jun kinase was dependent on Ras but independent of RalA, indicating divergent pathways activated by EGF and mediated by Ras. The transformed phenotype induced by EGF in the EGFR cells was dependent upon both Ras and RalA. Importantly, overexpression of wild-type RalA or an activated RalA mutant increased PLD activity in the absence of EGF and transformed the EGFR cells. Although overexpression of PLD1 is generally toxic to cells, the EGFR cells not only tolerated PLD1 overexpression but also became transformed in the absence of EGF. These data demonstrate that either RalA or PLD1 can cooperate with EGF receptor to transform cells.Overexpression of a tyrosine kinase is a common genetic defect in a variety of human tumors (21). The epidermal growth factor (EGF) receptor, which has an intrinsic tyrosine kinase that is activated in response to EGF, is frequently overexpressed in human breast and ovarian cancer (35). However, overexpression of a tyrosine kinase such as the EGF receptor is not sufficient for a fully transformed or cancerous phenotype. We recently demonstrated that downregulation of protein kinase C ␦ (PKC ␦) transforms 3Y1 rat fibroblasts overexpressing either c-Src (28) or the EGF receptor (19). The EGF receptor-overexpressing cells (EGFR cells) could also be transformed when treated with EGF (19), suggesting that EGF could accomplish what PKC ␦ downregulation accomplished. Interestingly, downregulation of PKC ␦ also caused an increase in phospholipase D (PLD) activity (19,38), which is commonly elevated in response to oncogenic and mitogenic stimuli (11,41). Both EGF-induced increases in PLD activity and EGFinduced transformation were dependent upon the ␣ isoform of PKC (19), suggesting that PLD may be an important component of the mitogenic and oncogenic properties of the EGF receptor.We demonstrated previously (30) that PLD1 associates directly with the small GTPase RalA, a downstream target of Ras (13). RalA is required for PLD activation in response to v-Src and v-Ras (22). RalA has also been implicated in cell transformation (1, 39), indicating a possible role for PLD in mitogenic signaling. In this paper, we report that both RalA and PLD1 can cooperate with an overexpressed EGF receptor to transform cells. MATERIALS AND METHODSCells and cell culture conditions. Rat 3Y1 cells or rat 3Y1 cells expressing the EGF receptor were maintained in Dulbecco's modified Eagle's medium supplemented with 10% bovine calf serum (HyClone) as described previously (28, 29). The EGF...

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Since TPA downregulates all phorbol ester-responsive PKC isoforms, we examined the effects of PKC ␦-and PKC ␣-specific inhibitors and the expression of dominant negative mutants for both PKC ␦ and ␣. Consistent with a tumor-suppressing function for PKC ␦, the PKC ␦-specific inhibitor rottlerin and a dominant negative PKC ␦ mutant transformed the EGFR cells in the absence of EGF. In contrast, the PKC ␣-specific inhibitor Go6976 and expression of a dominant negative PKC ␣ mutant blocked the transformed phenotype induced by both EGF and PKC ␦ inhibition. Interestingly, both rottlerin and EGF induced substantial increases in phospholipase D (PLD) activity, which is commonly elevated in response to mitogenic stimuli. The elevation of PLD activity in response to inhibiting PKC ␦, like transformation, was dependent upon PKC ␣ and restricted to the EGFR cells. These data demonstrate that PKC isoforms ␣ and ␦ have antagonistic effects on both transformation and PLD activity and further support a tumor suppressor role for PKC ␦ that may be mediated by suppression of tyrosine kinase-dependent increases in PLD activity.

A newly discovered interaction between LL5s, laminins, and integrins reveals how the extracellular matrix directs microtubule polarity in epithelial tissues.

It has been challenging to engineer lung adenocarcinoma models via oncogene-mediated transformation of primary cultured normal human cells. Although viral oncoprotein-mediated malignant transformation has been reported, xenografts derived from such transformed cells generally represent poorly differentiated cancers. Here, we demonstrate that the combined expression of multiple cellular factors induces malignant transformation in normal human lung epithelial cells. Although a combination of four genetic alterations, including hTERT overexpression, inactivation of the pRB and p53 pathways, and KRAS activation, is insufficient for normal human small airway epithelial cells to be fully transformed, expression of one additional oncogene induces malignant transformation. Notably, we have succeeded in reproducing human lung adenocarcinoma phenotypes in the flanks of nude mice by introducing an active form of PIK3CA, CYCLIN-D1, or a dominant-negative form of LKB1 in combination with the four genetic alterations above. Besides differentiated lung cancer, poorly differentiated cancer models can also be engineered by employing c-MYC as one of the genetic elements, indicating that histologic features and degree of differentiation of xenografts are controllable to some extent by changing the combination of genetic elements introduced. This is the first study reporting malignant transformation of normal lung epithelial cells in the absence of viral oncoproteins. We propose that our model system would be useful to identify the minimal and most crucial set of changes required for lung tumorigenesis, and that it would provide a broadly applicable approach for discovering attractive therapeutic targets. Cancer Res; 71(7);

Human diploid fibroblasts (HDF) immortalized by hTERT and simian virus 40 (SV40) early region (ER) exhibit a limited degree of transformation upon the expression of activated H-RAS (H-RAShuman diploid fibroblast ͉ mitogen-activated protein kinase kinase͞extracellular signal-regulated kinase ͉ simian virus 40 early region ͉ telomerase ͉ rat embryonic fibroblasts I n contrast to rodent cells, human cells have long been very difficult to be transformed in vitro. Normal (nonestablished) rodent cells can be readily transformed by the concomitant expression of two oncogenes, whereas normal human cells have proven to be resistant to the transformation induced by the same combinations of oncogenes, indicating the fundamental differences in cellular requirements for oncogenic transformation between these species. In 1999, Weinberg and colleagues (1) reported that ectopic expression of the catalytic subunit of human telomerase (hTERT) made normal human foreskin fibroblasts and kidney epithelial cells susceptible to transformation by the combined expression of simian virus 40 (SV40) early region (SV40 ER) and activated H-Ras (H-Ras V12). They further succeeded in transforming several different types of normal human cells by the same combinations of genetic elements. Based on these results, they proposed that the telomere maintenance by hTERT, inactivation of p53 and retinoblastoma (Rb) by SV40 large T antigen, suppression of protein phosphatase PP2A activity by SV40 small t antigen, and constitutive activation of mitogenic signaling by activated Ras are sufficient to transform normal human cells (2). However, we recently demonstrated that, even when all these requirements are fulfilled, the transformed phenotypes of human fibroblasts are much less malignant than those of rat fibroblasts in terms of morphological changes, anchorage independence, and tumorigenicity in nude mice (3, 4). Our results strongly suggest that normal human cells have still undefined intrinsic mechanisms rendering them resistant to oncogenic transformation. In the present study, we found that the expression of FRA1, a member of the family of AP-1 transcription factors, was differentially regulated by RAS in human and rat fibroblasts and concluded that this transcription factor is one of the determinant factors for species-specific susceptibility to RAS-induced transformation. Results FRA1 Is Highly Induced by RAS in Rodent Fibroblasts but Not in HumanFibroblasts. To explore the molecular basis underlying the difference in RAS-induced transformation phenotypes between rodent and human fibroblasts, we performed gene expression profiling by using a CodeLink microarray. Rat embryonic fibroblasts (REF), mouse embryonic fibroblasts (MEF) and a strain of human diploid fibroblasts (HDF), TIG3 ectopically expressing hTERT (TIG3͞T), were analyzed. We made comparisons between cells expressing SV40 ER (S) alone and cells coexpressing SV40 ER and H-Ras V12 (SR), because the drastic phenotypic differences between rodent and human fibroblasts were observed in respo...

The small GTPase Rho regulates cell morphogenesis through remodeling of the actin cytoskeleton. While Rho is overexpressed in many clinical cancers, the role of Rho signaling in oncogenesis remains unknown. mDia1 is a Rho effector producing straight actin filaments. Here we transduced mouse embryonic fibroblasts from mDia1-deficient mice with temperature-sensitive v-Src and examined the involvement and mechanism of the Rho-mDia1 pathway in Src-induced oncogenesis. We showed that in v-Src-transduced mDia1-deficient cells, formation of actin filaments is suppressed, and v-Src in the perinuclear region does not move to focal adhesions upon a temperature shift. Consequently, membrane translocation of v-Src, v-Src-induced morphological transformation, and podosome formation are all suppressed in mDia1-deficient cells with impaired tyrosine phosphorylation. mDia1-deficient cells show reduced transformation in vitro as examined by focus formation and colony formation in soft agar and exhibit suppressed tumorigenesis and invasion when implanted in nude mice in vivo. Given overexpression of c-Src in various cancers, these findings suggest that Rho-mDia1 signaling facilitates malignant transformation and invasion by manipulating the actin cytoskeleton and targeting Src to the cell periphery.

The c-abl tyrosine kinase is the proto-oncogene of the vabl oncogene of the Abelson murine leukemia virus. Although mutational variants of c-Abl can exhibit gain of function and can produce a transformed phenotype, the function of c-Abl in transformation remained unclear. Here, we report that the loss of c-abl facilitates transformation. c-abl-knockout mouse embryonic fibroblasts (MEFs) immortalized by SV40 T antigen acquired anchorage-independent growth, and by constructing mutational variants of T antigen we showed that binding of large T antigen to p53 and RB was necessary to induce anchorage-independent growth. Although c-abl/p53 double-knockout MEFs did not undergo anchorage-independent growth, those expressing human papilloma virus 16 E7, which mainly inactivates RB, did. Our results show that the loss of c-abl facilitates anchorage-independent growth in the context of p53 and RB deficiency, and suggest that loss of function of c-abl facilitates some types of transformation.