Loss of cell polarity drives tumor growth and invasion through JNK activation in Drosophila

Apparent defects in cell polarity are often seen in human cancer. However, the underlying mechanisms of how cell polarity disruption contributes to tumor progression are unknown. Here, using a Drosophila genetic model for Ras-induced tumor progression, we show a molecular link between loss of cell polarity and tumor malignancy. Mutation of different apicobasal polarity genes activates c-Jun N-terminal kinase (JNK) signaling and downregulates the E-cadherin/beta-catenin adhesion complex, both of which are necessary and sufficient to cause oncogenic Ras(V12)-induced benign tumors in the developing eye to exhibit metastatic behavior. Furthermore, activated JNK and Ras signaling cooperate in promoting tumor growth cell autonomously, as JNK signaling switches its proapoptotic role to a progrowth effect in the presence of oncogenic Ras. Our finding that such context-dependent alterations promote both tumor growth and metastatic behavior suggests that metastasis-promoting mutations may be selected for based primarily on their growth-promoting capabilities. Similar oncogenic cooperation mediated through these evolutionarily conserved signaling pathways could contribute to human cancer progression.     

Acquisition order of Ras and p53 gene alterations defines distinct adrenocortical tumor phenotypes

Sporadic adrenocortical carcinomas (ACC) are rare endocrine neoplasms with a dismal prognosis. By contrast, benign tumors of the adrenal cortex are common in the general population. Whether benign tumors represent a separate entity or are in fact part of a process of tumor progression ultimately leading to an ACC is still an unresolved issue. To this end, we have developed a mouse model of tumor progression by successively transducing genes altered in adrenocortical tumors into normal adrenocortical cells. The introduction in different orders of the oncogenic allele of Ras (H-Ras(G12V)) and the mutant p53(DD) that disrupts the p53 pathway yielded tumors displaying major differences in histological features, tumorigenicity, and metastatic behavior. Whereas the successive expression of Ras(G12V) and p53(DD) led to highly malignant tumors with metastatic behavior, reminiscent of those formed after the simultaneous introduction of p53(DD) and Ras(G12V), the reverse sequence gave rise only to benign tumors. Microarray profiling revealed that 157 genes related to cancer development and progression were differentially expressed. Of these genes, 40 were up-regulated and 117 were down-regulated in malignant cell populations as compared with benign cell populations. This is the first evidence-based observation that ACC development follows a multistage progression and that the tumor phenotype is directly influenced by the order of acquisition of genetic alterations.     

Brain cancer stem-like cell genesis from p53-deficient mouse astrocytes by oncogenic Ras

Here, we show that H-ras^V12 causes the p53-knockout mouse astrocytes (p53−/− astrocytes) to be transformed into brain cancer stem-like cells. H-ras^V12 triggers the p53−/− astrocytes to express a Nestin and a Cd133, which are expressed in normal and cancer neural stem cells. H-ras^V12 also induces the formation of a single cell-derived neurosphere under neural stem cell culture conditions. Furthermore, H-ras^V12-overexpressing p53−/− astrocytes (p53−/−ast-H-ras^V12) possess an in vitro self-renewal capacity, and are aberrantly differentiated into Tuj1-positve neurons both in vitro and in vivo. Amongst a variety of Ras-mediated canonical signaling pathways, we demonstrated that the MEK/ERK signaling pathway is responsible for neurosphere formation in p53-deficient astrocytes, whereas the PI3K/AKT signaling pathway is involved in oncogenic transformation in these cells. These findings suggest that the activation of Ras signaling pathways promotes the generation of brain cancer stem-like cells from p53-deficient mouse astrocytes by changing cell fate and transforming cell properties.     

Capns1, a new binding partner of RasGAP-SH3 domain in K-Ras(V12) oncogenic cells: modulation of cell survival and migration

Ras GTPase-activating protein (RasGAP) is hypothesized to be an effector of oncogenic Ras stimulating numerous downstream cellular signaling cascades involved in survival, proliferation and motility. In this study, we identified calpain small subunit-1 (Capns1) as a new RasGAP-SH3 domain binding partner, using yeast two-hybrid screening. The interaction was confirmed by co-immunoprecipitation assay and was found specific to cells expressing oncogenic K-Ras. We used confocal microscopy to analyze our stably transfected cell model producing mutant Ras (PC3Ras(V12)). Staining for RasGAP-SH3/Capns1 co-localization was two-fold stronger in the protrusions of Ras(V12) cells than in PC3 cells. RasGAP or Capns1 knockdown in PC3Ras(V12) cells induced a two- to three-fold increase in apoptosis. Capns1 gene silencing reduced the speed and increased the persistence of movement in PC3Ras(V12) cells. In contrast, RasGAP knockdown in PC3Ras(V12) cells increased cell migration. Knockdown of both proteins altered the speed and directionality of cell motility. Our findings suggest that RasGAP and Capns1 interaction in oncogenic Ras cells is involved in regulating migration and cell survival.     

Oncogenic Ras leads to Rho activation by activating the mitogen-activated protein kinase pathway and decreasing Rho-GTPase-activating protein activity

Transformation by oncogenic Ras requires signaling through Rho family proteins including RhoA, but the mechanism(s) whereby oncogenic Ras regulates the activity of RhoA is (are) unknown. We examined the effect of Ras on RhoA activity in NIH 3T3 cells either stably transfected with H-Ras(V12) under control of an inducible promoter or transiently expressing the activated H-Ras. Using a novel method to quantitate enzymatically the GTP bound to Rho, we found that expression of the oncogenic Ras increased Rho activity approximately 2-fold. Increased Rho activity was associated with increased plasma membrane binding of RhoA and decreased activity of the Rho/Ras-regulated p21(WAF1/CIP1) promoter. RhoA activation by oncogenic Ras could be explained by a decrease in cytosolic p190 Rho-GAP activity and translocation of p190 Rho-GAP from the cytosol to a detergent-insoluble cytoskeletal fraction. Pharmacologic inhibition of the Ras/Raf/MEK/ERK pathway prevented Ras-induced activation of RhoA and translocation of p190 Rho-GAP; expression of constitutively active Raf-1 kinase or MEK was sufficient to induce p190 Rho-GAP translocation. We conclude that in NIH 3T3 cells oncogenic Ras activates RhoA through the Raf/MEK/ERK pathway by decreasing the cytosolic activity and changing the subcellular localization of p190 Rho-GAP.     

p120 Catenin is required for growth factor-dependent cell motility and scattering in epithelial cells

Cadherin-mediated cell-cell adhesion is dynamically modulated during epithelial-mesenchymal transition triggered by activation of receptor tyrosine kinases (RTK) in epithelial cells. Several cadherin-binding proteins have been identified that control cell-cell adhesion. However, the mechanisms by which intercellular adhesion and cell motility are coregulated are still unknown. Here, we delineate a hitherto uncharted cooperation between RTKs, RhoA GTPase, and p120 catenin in instructing a motile behavior to epithelial cells. We found that expression of an N-terminus-deleted p120 catenin in a variety of epithelial cell types, including primary keratinocytes, effectively competes for endogenous p120 at cadherin binding sites and abrogates EGF-stimulated cell motility as well as HGF-induced cell scattering. The deleted mutant also inhibits the PI3K-dependent RhoA activation ensuing receptor activation. Conversely, we also show that the ectopic expression of full-length p120 in epithelial cells promotes cytoskeletal changes, stimulates cell motility, and activates RhoA. Both motogenic response to p120 and RhoA activation require coactivation of signaling downstream of RTKs as they are suppressed by ablation of the Ras/PI3K pathway. These studies demonstrate that p120 catenin is a necessary target of RTKs in regulating cell motility and help define a novel pathway leading to RhoA activation, which may contribute to the early steps of metastatic invasion.     

p27 as Jekyll and Hyde: Regulation of cell cycle and cell motility

p27 is a key regulator of cell proliferation. While it opposes cell cycle progression by binding to and inhibiting cyclin E-Cdk2, T157/198 phosphorylation of p27 promotes its assembly of D-type cyclin-CDKs. In addition to its actions on the cell cycle, p27 regulates CDK-independent cytoplasmic functions. In human cancers, oncogenic activation of the PI3K signaling pathway often results in cytoplasmic mislocalization of p27. Cytoplasmic p27 plays an important role in cell motility and migration; it binds and modulates activation of the RhoA/ROCK cascade. p27:RhoA binding is facilitated by p27 phosphorylation at threonine 198. Accumulation of cytoplasmic p27 leads to increased cellular motility, a critical event in tumor metastasis. Further characterization of post-translational modifications governing p27 localization and its action on RhoA and the actin cytoskeleton may provide critical insights into human cancer metastasis.     

Differential requirements for ras and the retinoblastoma tumor suppressor protein in the androgen dependence of prostatic adenocarcinoma cells.

Prostate cells are dependent on androgen for proliferation, but during tumor progression prostate cancer cells achieve independence from the androgen requirement. We report that androgen withdrawal fails to inhibit cell cycle progression or influence the expression of cyclin-dependent kinase (CDK)/cyclins in androgen-independent prostate cancer cells, indicating that these cells signal for cell cycle progression in the absence of androgen. However, phosphorylation of the retinoblastoma tumor suppressor protein (RB) is still required for G1-S progression in androgen-independent cells, since the expression of constitutively active RB (PSM-RB) or p16ink4a caused cell cycle arrest and mimicked the effects of androgen withdrawal on downstream targets in androgen-dependent LNCaP cells. Since Ras is known to mediate mitogenic signaling to RB, we hypothesized that active V12Ras would induce androgen-independent cell cycle progression in LNCaP cells. Although V12Ras was able to stimulate ERK phosphorylation and induce cyclin D1 expression in the absence of androgen, it was not sufficient to promote androgen-independent cell cycle progression. Similarly, ectopic expression of CDK4/cyclin D1, which stimulated RB phosphorylation in the presence of androgen, was incapable of inactivating RB or driving cell cycle progression in the absence of androgen. We show that androgen regulates both CDK4/cyclin D1 and CDK2 complexes to inactivate RB and initiate cell cycle progression. Together, these data show that androgen independence is achieved via deregulation of the androgen to RB signal, and that this signal can only be partially initiated by the Ras pathway in androgen-dependent cells.     

SmgGDS-558 regulates the cell cycle in pancreatic,non-small cell lung,and breast cancers

Oncogenic mutation or misregulation of small GTPases in the Ras and Rho families can promote unregulated cell cycle progression in cancer. Post-translational modification by prenylation of these GTPases allows them to signal at the cell membrane. Splice variants of SmgGDS, named SmgGDS-607 and SmgGDS-558, promote the prenylation and membrane trafficking of multiple Ras and Rho family members, which makes SmgGDS a potentially important regulator of the cell cycle. Surprisingly little is known about how SmgGDS-607 and SmgGDS-558 affect cell cycle-regulatory proteins in cancer, even though SmgGDS is overexpressed in multiple types of cancer. To examine the roles of SmgGDS splice variants in the cell cycle, we compared the effects of the RNAi-mediated depletion of SmgGDS-558 vs. SmgGDS-607 on cell cycle progression and the expression of cyclin D1, p27, and p21 in pancreatic, lung, and breast cancer cell lines. We show for the first time that SmgGDS promotes proliferation of pancreatic cancer cells, and we demonstrate that SmgGDS-558 plays a greater role than SmgGDS-607 in cell cycle progression as well as promoting cyclin D1 and suppressing p27 expression in multiple types of cancer. Silencing both splice variants of SmgGDS in the cancer cell lines produces an alternative signaling profile compared with silencing SmgGDS-558 alone. We also show that loss of both SmgGDS-607 and SmgGDS-558 simultaneously decreases tumorigenesis of NCI-H1703 non-small cell lung carcinoma (NSCLC) xenografts in mice. These findings indicate that SmgGDS promotes cell cycle progression in multiple types of cancer, making SmgGDS a valuable target for cancer therapeutics.     

p200 RhoGAP promotes cell proliferation by mediating cross-talk between Ras and Rho signaling pathways

p200 RhoGAP, a member of the Rho GTPase-activating protein (RhoGAP) family, was previously implicated in the regulation of neurite outgrowth through its RhoGAP activity. Here we show that ectopic expression of p200 RhoGAP stimulates fibroblast cell proliferation and cell cycle progression, leading to transformation. The morphology of the foci induced by p200 RhoGAP is distinct from that formed by Rac or Rho activation but similar to that induced by oncogenic Ras, raising the possibility that p200 RhoGAP may engage Ras signaling. Expression of p200 RhoGAP results in a significant increase of Ras-GTP and the activation of two downstream signaling pathways of Ras, ERK1/2 and phosphatidylinositol 3-kinase. Inhibition of Ras or ERK1/2, but not phosphatidylinositol 3-kinase, effectively suppresses the foci formation induced by p200 RhoGAP, suggesting that the Ras-ERK pathway is required for p200 RhoGAP-mediated cell transformation. p200 RhoGAP co-localizes with p120 RasGAP in cells and forms a complex with p120 RasGAP, and this interaction is mediated by the C-terminal region and the Src homology 3 domain of p200 RhoGAP and p120 RasGAP, respectively. Mutations of p200 RhoGAP that disrupt interaction with p120 RasGAP abolish its Ras activation and cell transforming activities. Interestingly, the RhoGAP activity of the N-terminal RhoGAP domain in p200 RhoGAP is also required for its full transforming activity, and expression of a dominant negative RhoA mutant that blocks RhoA cycling between the GDP- and GTP-bound states suppresses p200 RhoGAP transformation. These results suggest that a Rho GTPase-activating protein may have a positive input to cell proliferation and provide evidence that p200 RhoGAP can mediate cross-talks between Ras- and Rho-regulated signaling pathways in cell growth regulation.     

Ras-Raf-Arf signaling critically depends on the Dmp1 transcription factor

Dmp1 prevents tumor formation by activating the Arf-p53 pathway. In cultured primary cells, the Dmp1 promoter was efficiently activated by oncogenic Ha-Ras(V12), but not by overexpressed c-Myc or E2F-1. Dmp1 promoter activation by Ras(V12) depended on Raf-MEK-ERK signaling. Induction of p19(Arf) and p21(Cip1) by oncogenic Raf was compromised in Dmp1-null cells, which were resistant to Raf-mediated premature senescence. A Ras(V12)-responsive element was mapped to the 5' leader sequence of the murine Dmp1 promoter, where endogenous Fos and Jun family proteins bind. Dmp1 promoter activation by Ras(V12) was strikingly impaired in c-Jun as well as in JunB knock-down cells, suggesting the critical role of Jun proteins in the activation of the Dmp1 promoter. A Ras(V12)-responsive element was mapped to the unique Dmp1/Ets site on the Arf promoter, where endogenous Dmp1 proteins bind upon oncogenic Raf activation. Therefore, activation of Arf by Ras/Raf signaling is indirectly mediated by Dmp1, explaining why Dmp1-null primary cells are highly susceptible to Ras-induced transformation. Our data indicate the presence of the novel Jun-Dmp1 pathway that directly links oncogenic Ras-Raf signaling and p19(Arf), independent of the classical cyclin D1/Cdk4-Rb-E2F pathway.     

Dissecting the contribution of p16(INK4A) and the Rb family to the Ras transformed phenotype

Although oncogenic Ras commonly contributes to the development of cancer, in normal primary cells it induces cell cycle arrest rather than transformation. Here we analyze the additional genetic changes required for Ras to promote cell cycle progression rather than arrest. We show that loss of p53 is sufficient for oncogenic Ras to stimulate proliferation in the absence of extrinsic mitogens in attached cells. However, surprisingly, we find that p53 loss is not sufficient for Ras to overcome anchorage dependence or contact inhibition. In contrast, expression of simian virus 40 (SV40) large T antigen (LT) allows Ras to overcome these additional cell cycle controls. Mutational analysis of SV40 LT shows that this action of SV40 LT depends on its ability to inactivate the retinoblastoma (Rb) family of proteins, in concert with the loss of p53. Importantly, we show that inactivation of the Rb family of proteins can be mimicked by loss of the cyclin-dependent kinase inhibitor p16(INK4A). p16(INK4A) is commonly lost in human tumors, but its contribution to the transformed phenotype is unknown. We demonstrate here a role for p16(INK4A) in the loss of cell cycle controls required for tumorigenesis and show how accumulating genetic changes cooperate and contribute to the transformed phenotype.     

Activated Ras induces cytoplasmic vacuolation and non-apoptotic death in glioblastoma cells via novel effector pathways

Expression of activated H-Ras induces a unique form of non-apoptotic cell death in human glioblastoma cells and other specific tumor cell lines. The major cytopathological features of this form of death are the accumulation of large phase-lucent, LAMP1-positive, cytoplasmic vacuoles. In this study we sought to determine if induction of cytoplasmic vacuolation a) depends on Ras farnesylation, b) is specific to H-Ras, and c) is mediated by signaling through the major known Ras effector pathways. We find that the unusual effects of activated H-Ras depend on farnesylation and membrane association of the GTPase. Both H-Ras(G12V) and K-Ras4B(G12V) stimulate vacuolation, but activated forms of Cdc42 and RhoA do not. Amino acid substitutions in the Ras effector domain, which are known to selectively impair its interactions with Raf kinase, class-I phosphatidylinositide 3-kinase (PI3K), or Ral nucleotide exchange factors, initially pointed to Raf as a possible mediator of cell vacuolation. However, the MEK inhibitor, PD98059, did not block the induction of vacuoles, and constitutively active Raf-Caax did not mimic the effects of Ras(G12V). Introduction of normal PTEN together with H-Ras(G12V) into U251 glioblastoma cells reduced the PI3K-dependent activation of Akt, but had no effect on vacuolation. Finally, co-expression of H-Ras(G12V) with a dominant-negative form of RalA did not suppress vacuolation. Taken together, the observations indicate that Ras activates non-conventional and perhaps unique effector pathways to induce cytoplasmic vacuolation in glioblastoma cells. Identification of the relevant signaling pathways may uncover specific molecular targets that can be manipulated to activate non-apoptotic cell death in this type of cancer.     

Dissecting the senescence-like program in tumor cells activated by Ras signaling

Activated Ras signaling can induce a permanent growth arrest in osteosarcoma cells. Here, we report that a senescence-like growth inhibition is also achieved in human carcinoma cells upon the transduction of H-Ras(V12). Ras-induced tumor senescence can be recapitulated by the transduction of activated, but not wild-type, MEK. The ability for H-Ras(V12) to suppress tumor cell growth is drastically compromised in cells that harbor endogenous activating ras mutations. Notably, growth inhibition of tumor cells containing ras mutations can be achieved through the introduction of activated MEK. Tumor senescence induced by Ras signaling can occur in the absence of p16 or Rb and is not interrupted by the inactivation of Rb, p107, or p130 via short hairpin RNA or the transduction with HPV16 E7. In contrast, inactivation of p21 via short hairpin RNA disrupts Ras-induced tumor senescence. In summary, this study uncovers a senescence-like program activated by Ras signaling to inhibit cancer cell growth. This program appears to be intact in cancer cells that do not harbor ras mutations. Moreover, cancer cells that carry ras mutations remain susceptible to tumor senescence induced by activated MEK. These novel findings can potentially lead to the development of innovative cancer intervention.     

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.     

p120 catenin induces opposing effects on tumor cell growth depending on E-cadherin expression

p120 catenin regulates the activity of the Rho family guanosine triphosphatases (including RhoA and Rac1) in an adhesion-dependent manner. Through this action, p120 promotes a sessile cellular phenotype when associated with epithelial cadherin (E-cadherin) or a motile phenotype when associated with mesenchymal cadherins. In this study, we show that p120 also exerts significant and diametrically opposing effects on tumor cell growth depending on E-cadherin expression. Endogenous p120 acts to stabilize E-cadherin complexes and to actively promote the tumor-suppressive function of E-cadherin, potently inhibiting Ras activation. Upon E-cadherin loss during tumor progression, the negative regulation of Ras is relieved; under these conditions, endogenous p120 promotes transformed cell growth both in vitro and in vivo by activating a Rac1–mitogen-activated protein kinase signaling pathway normally activated by the adhesion of cells to the extracellular matrix. These data indicate that both E-cadherin and p120 are important regulators of tumor cell growth and imply roles for both proteins in chemoresistance and targeted therapeutics.     

The PDZ binding motif of human papillomavirus type 16 E6 induces PTPN13 loss, which allows anchorage-independent growth and synergizes with ras for invasive growth

The human papillomavirus (HPV) oncogene E6 has been shown to perform multiple functions (p53 degradation, telomerase activation, etc.) that play a role in oncogenic transformation. Beyond known E6 functions, an undefined mechanism that allows cellular invasion requires the E6 PDZ binding motif (PDZBM). Here, we show that HPV type 16 (HPV16) E6 interacts with and induces loss of a protein tyrosine phosphatase (PTPN13) in a PDZBM-dependent manner. PTPN13 loss induced either by the presence of E6 or by a short hairpin RNA strategy allows for anchorage-independent growth (AIG) and synergy with a known oncogene, Ras^v12, resulting in invasive growth in vivo. Restoring PTPN13 expression reverses AIG in cells lacking PTPN13. A genomic analysis of colorectal carcinoma has identified an association between PTPN13 loss-of-function mutations and aberrant Ras signaling. Our findings support this correlation and provide methods for further evaluation of the mechanisms by which PTPN13 loss/Ras expression leads to invasive growth, the results of which will be important for treatment of HPV-related and non-HPV cancer.