Klaudia Giehl

Oncogenic Ras in tumour progression and metastasis

Abstract The ras genes give rise to a family of related GTP-binding proteins that exhibit potent transforming potential. Mutational activation of Ras proteins promotes oncogenesis by disturbing a multitude of cellular processes, such as gene expression, cell cycle progression and cell proliferation, as well as cell survival, and cell migration. Ras signalling pathways are well known for their involvement in tumour initiation, but less is known about their contribution to invasion and metastasis. This review summarises the role and mechanisms of Ras signalling, especially the role of the Ras effector cascade Raf/MEK/ERK, as well as the phosphatidylinositol 3-kinase/Akt pathway in Ras-mediated transformation and tumour progression. In addition, it discusses the impact of Rho GTPases on Ras-mediated transformation and metastasis.

Paraxial protocadherin coordinates cell polarity during convergent extension via Rho A and JNK

Convergent extension movements occur ubiquitously in animal development. This special type of cell movement is controlled by the Wnt/planar cell polarity (PCP) pathway. Here we show that Xenopus paraxial protocadherin (XPAPC) functionally interacts with the Wnt/PCP pathway in the control of convergence and extension (CE) movements in Xenopus laevis. XPAPC functions as a signalling molecule that coordinates cell polarity of the involuting mesoderm in mediolateral orientation and thus selectively promotes convergence in CE movements. XPAPC signals through the small GTPases Rho A and Rac 1 and c‐jun N‐terminal kinase (JNK). Loss of XPAPC function blocks Rho A‐mediated JNK activation. Despite common downstream components, XPAPC and Wnt/PCP signalling are not redundant, and the activity of both, XPAPC and PCP signalling, is required to coordinate CE movements.

Mechanisms in LPA-induced tumor cell migration: critical role of phosphorylated ERK

Lysophosphatidic acid (LPA) is a serum-borne phospholipid with hormone and growth factor-like properties. LPA has been shown to modulate tumor cell invasion and malignant cell growth. Here, we report that two human pancreatic carcinoma cell lines, PANC-1 and BxPC-3, express functionally active LPA receptors coupled to pertussis toxin-sensitive Gi/o-proteins. In contrast to other cell types, LPA does not act as a mitogen, but is an efficacious stimulator of cell migration of these tumor cells. LPA-induced chemotaxis is markedly dependent on activation of PTX-sensitive heterotrimeric G-proteins, on activation of the small GTPases Ras, Rac and RhoA, and on GTPase-dependent activation of ERK. LPA-induced ERK activation results in a transient translocation of the phosphorylated ERK to newly forming focal contact sites at the leading edge of the migrating cells. Inhibition of ERK activation and its subsequent translocation impaired LPA-induced chemotaxis and LPA-induced actin reorganization. Thus, pancreatic tumor cell migration in response to LPA is essentially controlled by activation of a Gi/o-ERK pathway and requires the LPA-induced activation of Ras, Rac1 and RhoA.

TGFβ-induced downregulation of E-cadherin-based cell-cell adhesion depends on PI3-kinase and PTEN

Transforming growth factor beta (TGFβ) has profound growth-suppressive effects on normal epithelial cells, but supports metastasis formation in many tumour types. In most epithelial tumour cells TGFβ1 treatment results in epithelial dedifferentiation with reduced cell aggregation and enhanced cellular migration. Here we show that the epithelial dedifferentiation, accompanied by dissociation of the E-cadherin adhesion complex, induced by TGFβ1 depended on phosphatidylinositol 3-kinase (PI3-kinase) and the phosphatase PTEN as analysed in PANC-1 and Smad4-deficient BxPC-3 pancreatic carcinoma cells. TGFβ1 treatment enhanced tyrosine phosphorylation of α- and β-catenin, which resulted in dissociation of the E-cadherin/catenin complex from the actin cytoskeleton and reduced cell-cell adhesion. The PI3-kinase and PTEN were found associated with the E-cadherin/catenin complex via β-catenin. TGFβ1 treatment reduced the amount of PTEN bound to β-catenin and markedly increased the tyrosine phosphorylation of β-catenin. By contrast, forced expression of PTEN clearly reduced the TGFβ1-induced phosphorylation of β-catenin. The TGFβ1-induced β-catenin phosphorylation was also dependent on PI3-kinase and Ras activity. The described effects of TGFβ1 were independent of Smad4, which is homozygous deleted in BxPC-3 cells. Collectively, these data show that the TGFβ1-induced destabilisation of E-cadherin-mediated cell-cell adhesion involves phosphorylation of β-catenin, which is regulated by E-cadherin adhesion complex-associated PI3-kinase and PTEN.

The Role of Metastasis-Associated Protein 1 in Prostate Cancer Progression

Distinguishing aggressive prostate cancer from indolent disease represents an important clinical challenge, as current therapy requires over treating men with prostate cancer to prevent the progression of a few cases. Expression of the metastasis-associated protein 1 (MTA1) has previously been found to be associated with progression to the metastatic state in various cancers. Analyzing DNA microarray data, we found MTA1 to be selectively overexpressed in metastatic prostate cancer compared with clinically localized prostate cancer and benign prostate tissue. These results were validated by demonstrating overexpression of MTA1 in metastatic prostate cancer by immunoblot analysis. MTA1 protein expression was evaluated by immunohistochemistry in a broad spectrum of prostate tumors with tissue microarrays containing 1940 tissue cores from 300 cases. Metastatic prostate cancer demonstrated significantly higher mean MTA1 protein expression intensity (score = 3.4/4) and percentage of tissue cores staining positive for MTA1 (83%) compared with clinically localized prostate cancer (score = 2.8/4, 63% positive cores) or benign prostate tissue (score = 1.5/4, 25% positive cores) with a mean difference of 0.54 and 1.84, respectively (P < 0.00001 for both). Paradoxically, for localized disease, higher MTA1 protein expression was associated with lower rates of prostate specific antigen recurrence after radical prostatectomy for localized disease. In summary, this study identified an association of MTA1 expression and prostate cancer progression.

Activation of Phosphatidylinositol 3-Kinase and Extracellular Signal-Regulated Kinase Is Required for Glial Cell Line-Derived Neurotrophic Factor-Induced Migration and Invasion of Pancreatic Carcinoma Cells

Pancreatic carcinoma cells exhibit a pronounced tendency to invade along and into intra- and extrapancreatic nerves, even at early stages of the disease. The neurotrophic factor glial cell line-derived neurotrophic factor (GDNF) has been shown to promote pancreatic cancer cell invasion. Here, we demonstrate that pancreatic carcinoma cell lines, such as PANC-1, expressed the RET and GDNF family receptor α receptor components for GDNF and that primary pancreatic tumor samples, derived from carcinomas with regional lymph node metastasis, exhibited marked expression of the mRNA encoding the RET51 isoform. Moreover, GDNF was an efficacious and potent chemoattractant for pancreatic carcinoma cells as examined in in vitro and in vivo model systems. Treatment of PANC-1 cells with GDNF resulted in activation of the monomeric GTPases N-Ras, Rac1, and RhoA, in activation of the mitogen-activated protein kinases extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) and in activation of the phosphatidylinositol 3-kinase/Akt pathway. Both inhibition of the Ras-Raf-MEK (mitogen-activated protein/ERK kinase)-ERK cascade by either stable expression of dominant-negative H-Ras(N17) or addition of the MEK1 inhibitor PD98059 as well as inhibition of the phosphatidylinositol 3-kinase pathway by LY294002 prevented GDNF-induced migration and invasion of PANC-1 cells. These results demonstrate that pancreatic tumor cell migration and possibly perineural invasion in response to GDNF is critically controlled by activation of the Ras-Raf-MEK-ERK and the phosphatidylinositol 3-kinase pathway.

A new functional Ras antagonist inhibits human pancreatic tumor growth in nude mice

Constitutively active Ras proteins, their regulatory components, and overexpressed tyrosine kinase receptors that activate Ras, are frequently associated with cell transformation in human tumors. This suggests that functional Ras antagonists may have anti-tumor activity. Studies in rodent fibroblasts have shown that S-trans, transfarnesylthiosalicylic acid (FTS) acts as a rather specific nontoxic Ras antagonist, dislodging Ras from its membrane anchorage domains and accelerating its degradation. FTS is not a farnesyltransferase inhibitor, and does not affect Ras maturation. Here we demonstrate that FTS also acts as a functional Ras antagonist in human pancreatic cell lines that express activated K-Ras (Panc-1 and MiaPaCa-2). In Panc-1 cells, FTS at a concentration of 25 – 100 μM reduced the amount of Ras in a dose-dependent manner and interfered with serum-dependent and epidermal growth factor-stimulated ERK activation, thus inhibiting both anchorage-dependent and anchorage-independent growth of Panc-1 cells in vitro. FTS also inhibited tumor growth in Panc-1 xenografted nude mice, apparently without systemic toxicity. Daily FTS treatment (5 mg/kg intraperitoneally) in mice with tumors (mean volume 0.07 cm3) markedly decreased tumor growth (after treatment for 18 days, tumor volume had increased by only 23±30-fold in the FTS-treated group and by 127±66-fold in controls). These findings suggest that FTS represents a new class of functional Ras antagonists with potential therapeutic value.

Growth factor-dependent activation of the Ras-Raf-MEK-MAPK pathway in the human pancreatic carcinoma cell line PANC-1 carrying activated K-ras: implications for cell proliferation and cell migration.

Human ductal adenocarcinoma of the pancreas frequently carry activating point mutations in the K-ras protooncogene. We have analysed the activity of the Ras-Raf-MEK-MAPK cascade in the human pancreatic carcinoma cell line PANC-1 carrying an activating K-ras mutation. Serum-starved cells and cells grown in medium with serum did not show constitutively activated c-Raf, MEK-1, or p42 MAPK. Stimulation of cells with epidermal growth factor (EGF) or fetal calf serum (FCS) resulted in activation of N-Ras, but not K-Ras, as well as activation of c-Raf, MEK-1, and p42 MAPK. Preincubation of serum-starved cells with MEK-1 inhibitor PD98059 abolished EGF- and FCS-induced MAPK activation, identifying MEK as the upstream activator of MAPK. PANC-1 cells exhibited marked serum-dependence of anchorage-dependent and -independent cell growth as well as cell migration. EGF, alone or in combination with insulin and transferrin, did not induce cell proliferation of serum-starved PANC-1 cells, indicating that activation of MAPK alone was not sufficient to induce cell proliferation. FCS-induced DNA synthesis was inhibited by 40% by the MEK-1 inhibitor. On the other hand, treatment with either FCS or EGF alone resulted in marked, MEK-dependent increase of directed cell migration. Collectively, our results show that the activating K-ras mutation in PANC-1 cells does not result in constitutively increased Raf-MEK-MAPK signaling. Signal transduction via the Ras-Raf-MEK-MAPK cascade is maintained in these cells and is required for growth factor-induced cell proliferation and directed cell migration.

Modulation of Calcium-Activated Potassium Channels Induces Cardiogenesis of Pluripotent Stem Cells and Enrichment of Pacemaker-Like Cells

Background— Ion channels are key determinants for the function of excitable cells, but little is known about their role and involvement during cardiac development. Earlier work identified Ca2+-activated potassium channels of small and intermediate conductance (SKCas) as important regulators of neural stem cell fate. Here we have investigated their impact on the differentiation of pluripotent cells toward the cardiac lineage. Methods and Results— We have applied the SKCa activator 1-ethyl-2-benzimidazolinone on embryonic stem cells and identified this particular ion channel family as a new critical target involved in the generation of cardiac pacemaker-like cells: SKCa activation led to rapid remodeling of the actin cytoskeleton, inhibition of proliferation, induction of differentiation, and diminished teratoma formation. Time-restricted SKCa activation induced cardiac mesoderm and commitment to the cardiac lineage as shown by gene regulation, protein, and functional electrophysiological studies. In addition, the differentiation into cardiomyocytes was modulated in a qualitative fashion, resulting in a strong enrichment of pacemaker-like cells. This was accompanied by induction of the sino-atrial gene program and in parallel by a loss of the chamber-specific myocardium. In addition, SKCa activity induced activation of the Ras-Mek-Erk signaling cascade, a signaling pathway involved in the 1-ethyl-2-benzimidazolinone–induced effects. Conclusions— SKCa activation drives the fate of pluripotent cells toward mesoderm commitment and cardiomyocyte specification, preferentially into nodal-like cardiomyocytes. This provides a novel strategy for the enrichment of cardiomyocytes and in particular, the generation of a specific subtype of cardiomyocytes, pacemaker-like cells, without genetic modification.

TGFβ1 represses proliferation of pancreatic carcinoma cells which correlates with Smad4-independent inhibition of ERK activation

Transforming growth factor beta (TGFβ) is a tumor suppressor acting as inhibitor of cell cycle progression of epithelial cells. We show that treatment of the pancreatic carcinoma cell lines PANC-1 and BxPC-3 with TGFβ1 inhibits both growth factor-induced activation of the extracellular signal-regulated kinase 2 (ERK2) and translocation of the kinase to the nucleus. TGFβ1 causes a concentration-dependent reduction of cell proliferation in both cell lines. By measuring ERK activation, we can show that TGFβ1 is able to repress ERK activation induced by mitogenic stimuli such as EGF. This inhibitory effect of TGFβ1 is not mediated by suppression of Ras or c-Raf-1 activation, but mediated by TGFβ1-induced activation of a serine-threonine phosphatase, as demonstrated by inhibition of phosphatases by treatment with okadaic acid. Results obtained in the Smad4-deficient pancreatic carcinoma cell line BxPC-3, demonstrate that TGFβ1-induced growth inhibition is mediated by a Smad4-independent prevention of ERK2 activation. In contrast to the effects of TGFβ1 on epithelial cells, mesenchymal NIH3T3 fibroblasts exhibit elevated ERK2 activation and increased cell proliferation in response to TGFβ1 treatment. Smad4-independent phosphatase-mediated inhibition of mitogen-activated ERK2 represents a novel effector pathway contributing to suppression of epithelial pancreatic carcinoma cell proliferation by TGFβ1, in addition to the well-known Smad-induced tumor suppressor activity of TGFβ.