Kras oncogene analysis of non-melanoma skin tumors in Southeastern Brazil

Skin cancers are the most common human malignant neoplasia and their incidence is growing, chiefly in tropical countries. There is evidence that ultraviolet (UV) radiation present in sunlight is important for genetic damage. Mutations due to such damage could be responsible for alterations in oncogenes and tumor suppressor genes. Recent studies have reported remarkable differences in mutation frequency of the RAS proto-oncogene in non-melanoma skin cancers. These findings may reflect differences in the molecular epidemiology of cutaneous tumors found in geographical areas with diverse sun exposure and ethnical origins of their populations. Our study proposed to perform molecular analyses of skin tumors on patients living in southeastern Brazil, in areas with high levels of sun exposure. DNA from eight solar keratose (SK), 26 basal cell carcinomas (BCC) and 19 squamous cell carcinomas (SCC) was submitted to PCR-SSCP analysis for codons 12, 13 and 61. Contradicting other authors, we found no mutations in codons 12,13 but detected two BCCs and one SCC with a mutation in codon 61. These findings suggest that the activation of KRAS oncogene may contribute to the pathogenicity of cutaneous lesions in southeastern Brazil.

Metastatic lung cancer in a new mouse model inheriting p53 and latent K-ras mutations

Lung cancer is a devastating disease with very poor prognosis. The design of better treatments for patients would be greatly aided by mouse models that closely resemble the human disease. The most common type of human lung cancer is adenocarcinoma with frequent metastasis. Unfortunately, current models for this tumor are inadequate due to the absence of metastasis. Based on the molecular findings in human lung cancer and metastatic potential of osteosarcomas in mutant p53 mouse models, I hypothesized that mice with both K-ras and p53 missense mutations might develop metastatic lung adenocarcinomas. Therefore, I incorporated both K-rasLA1 and p53RI72HΔg alleles into mouse lung cells to establish a more faithful model for human lung adenocarcinoma and for translational and mechanistic studies. Mice with both mutations ( K-rasLA1/+ p53R172HΔg/+) developed advanced lung adenocarcinomas with similar histopathology to human tumors. These lung adenocarcinomas were highly aggressive and metastasized to multiple intrathoracic and extrathoracic sites in a pattern similar to that seen in lung cancer patients. This mouse model also showed gender differences in cancer related death and developed pleural mesotheliomas in 23.2% of them. In a preclinical study, the new drug Erlotinib (Tarceva) decreased the number and size of lung lesions in this model. These data demonstrate that this mouse model most closely mimics human metastatic lung adenocarcinoma and provides an invaluable system for translational studies. ^ To screen for important genes for metastasis, gene expression profiles of primary lung adenocarcinomas and metastases were analyzed. Microarray data showed that these two groups were segregated in gene expression and had 79 highly differentially expressed genes (more than 2.5 fold changes and p<0.001). Microarray data of Bub1b, Vimentin and CCAM1 were validated in tumors by quantitative real-time PCR (QPCR). Bub1b , a mitotic checkpoint gene, was overexpressed in metastases and this correlated with more chromosomal abnormalities in metastatic cells. Vimentin, a marker of epithelial-mesenchymal transition (EMT), was also highly expressed in metastases. Interestingly, Twist, a key EMT inducer, was also highly upregulated in metastases by QPCR, and this significantly correlated with the overexpression of Vimentin in the same tumors. These data suggest EMT occurs in lung adenocarcinomas and is a key mechanism for the development of metastasis in K-ras LA1/+ p53R172HΔg/+ mice. Thus, this mouse model provides a unique system to further probe the molecular basis of metastatic lung cancer.^

Increased geranylgeranylated K-Ras contributes to antineoplastic effects of farnesyltransferase inhibitors.

The Ras family of small GTPases (N-, H-, and K-Ras) is a group of important signaling mediators. Ras is frequently activated in some cancers, while others maintain low level activity to achieve optimal cell growth. In cells with endogenously low levels of active Ras, increasing Ras signaling through the ERK and p38 MAPK pathways can cause growth arrest or cell death. Ras requires prenylation – the addition of a 15-carbon (farnesyl) or 20-carbon (geranylgeranyl) group – to keep the protein anchored into membranes for effective signaling. N- and K-Ras can be alternatively geranylgeranylated (GG’d) if farnesylation is inhibited but are preferentially farnesylated. Small molecule inhibitors of farnesyltransferase (FTIs) have been developed as a means to alter Ras signaling. Our initial studies with FTIs in malignant and non-malignant cells revealed FTI-induced cell cycle arrest, reduced proliferation, and increased Ras signaling. These findings led us to the hypothesis that FTI induced increased GG’d Ras. We further hypothesized that the specific effects of FTI on cell cycle and growth result from increased signal strength of GG’d Ras. Our results did show that increase in GG’d K-Ras in particular results in reduced cell viability and cell cycle arrest. Genetically engineered constructs capable of only one type of prenylation confirmed that GG’d K-Ras recapitulated the effect of FTI in 293T cells. In tumor cell lines ERK and p38 MAPK pathways were both strongly activated in response to FTI, indicating the increased activity of GG’d K-Ras results in antiproliferative signals specifically through these pathways. These results collectively indicate FTI increases active GG’d K-Ras which activates ERK and p38 MAPKs to reduced cell viability and induce cell cycle arrest in malignant cells. This is the first report that identifies increased activity of GG’d K-Ras contributes to antineoplastic effects from FTI by increasing the activity of downstream MAPKs. Our observations suggest increased GG’d K-Ras activity, rather than inhibition of farnesylated Ras, is a major source of the cytostatic and cytotoxic effects of FTI. Our data may allow for determination of which patients would benefit from FTI by excluding tumors or diseases which have strong K-Ras signaling.

Acceleration of the PanIN Development in Mice Expressing Oncogenic K-Ras Due to a High Fat Diet

Obesity is postulated to be one of the major risk factors for pancreatic cancer, and recently it was indicated that an elevated body mass index (BMI correlates strongly with a decrease in patient survival. Despite the evident relationship, the molecular mechanisms involved are unclear. Oncogenic mutation of K-Ras is found early and is universal in pancreatic cancer. Extensive evidence indicates oncogenic K-Ras is not entirely active and it requires a triggering event to surpass the activity of Ras beyond the threshold necessary for a Ras-inflammation feed-forward loop. We hypothesize that high fat intake induces a persistent low level inflammatory response triggering increased K-Ras activity and that Cox-2 is essential for this inflammatory reaction. To determine this, LSL-K-Ras mice were crossed with Ela-CreER (Acinar-specific) or Pdx-1-Cre (Pancreas-specific) to “knock-in” oncogenic K-Ras. Additionally, these animals were crossed with Cox-2 conditional knockout mice to access the importance of Cox-2 in the inflammatory loop present. The mice were fed isocaloric diets containing 60% energy or 10% energy from fat. We found that a high fat diet increased K-Ras activity, PanIN formation, and fibrotic stroma significantly compared to a control diet. Genetic deletion of Cox-2 prevented high fat diet induced fibrosis and PanIN formation in oncogenic K-Ras expressing mice. Additionally, long term consumption of high fat diet, increased the progression of PanIN lesions leading to invasive cancer and decreased overall survival rate. These findings indicate that a high fat diet can stimulate the activation of oncogenic K-Ras and initiate an inflammatory feed forward loop requiring Cox-2 leading to inflammation, fibrosis, and PanINs. This mechanism could explain the relationship between a high fat diet and elevated risk for pancreatic cancer.

CONFORMATIONAL DYNAMICS OF K-RAS AND H-RAS PROTEINS: IS THERE FUNCTIONAL SPECIFICITY AT THE CATALYTIC DOMAIN?

Ras proteins serve as crucial signaling modulators in cell proliferation through their ability to hydrolyze GTP and exist in a GTP “on” state and GTP “off” state. There are three different human Ras isoforms: H-ras, N-ras and K-ras (4A and 4B). Although their sequence identity is very high at the catalytic domain, these isoforms differ in their ability to activate different effectors and hence different signaling pathways. Much of the previous work on this topic has attributed this difference to the hyper variable region of Ras proteins, which contains most of the sequence variance among the isoforms and encodes specificity for differential distribution in the membrane. However, we hypothesize that sequence variation on lobe II of Ras catalytic domain alters dynamics and leads to differential preference for different effectors or modulators. In this work, we used all atom molecular dynamics to analyze the dynamics in the catalytic domain of H-ras and K-ras. We have also analyzed the dynamics of a transforming mutant of H-ras and K-ras and further studied the dynamics of an effectorselective mutant of H-ras. Collectively we have determined that wild type K-ras is more dynamic than H-ras and that the structure of the effector binding loop more closely resembles that of the T35S Raf-selective mutant, possibly giving us a new view and insight into the v mode of effector specificity. Furthermore we have determined that specific mutations at the same location perturb the conformational equilibrium differently in H-ras and K-ras and that an enhanced oncogenic potential may arise from different structural perturbations for each point mutation of a specific isoform.

H-ras, K-ras, and inner plasma membrane raft proteins operate in nanoclusters with differential dependence on the actin cytoskeleton

Plasma membrane compartmentalization imposes lateral segregation on membrane proteins that is important for regulating signal transduction. We use computational modeling of immunogold spatial point patterns on intact plasma membrane sheets to test different models of inner plasma membrane organization. We find compartmentalization at the nanoscale level but show that a classical raft model of preexisting stable domains into which lipid raft proteins partition is incompatible with the spatial point patterns generated by the immunogold labeling of a palmitoylated raft marker protein. Rather, approximate to 30% of the raft protein exists in cholesterol-dependent nanoclusters, with approximate to 70% distributed as monomers. The cluster/monomer ratio (number of proteins in clusters/number of proteins outside clusters) is independent of expression level. H-rasG12V and K-rasG12V proteins also operate in nanoclusters with fixed cluster/monomer ratios that are independent of expression level. Detailed calibration of the immunogold imaging protocol suggests that radii of raft and RasG12V protein nanoclusters may be as small as 11 and 6 nm, respectively, and shows that the nanoclusters contain small numbers (6.0-7.7) of proteins. Raft nanoclusters do not form if the actin cytoskeleton is disassembled. The formation of K-rasG12V but not H-rasG12V nanoclusters also is actin-dependent. K-rasG12V but not H-rasG12V signaling is abrogated by actin cytoskeleton disassembly, which shows that nanoclustering is critical for Ras function. These findings argue against stable preexisting domains on the inner plasma membrane in favor of dynamic actively regulated nanoclusters similar to those proposed for the outer plasma membrane. RasG12V nanoclusters may facilitate the assembly of essential signal transduction complexes.

3531: Neoadjuvant radiotherapy (RT) combined with capecitabine (Cape) and sorafenib (Sor) in patients (pts) with locally advanced, k-ras-mutated rectal cancer (LARC): A phase I/II trial SAKK 41/08

Background: K-ras mutation is found in up to 40% of LARC. Sor is a multitarget tyrosine kinase inhibitor including raf and VEGFR and has demonstrated radiosensitizing effects. Sor might improve outcome of standard preoperative radio-chemotherapy in patients with k-ras mutated LARC. Methods: Pts with k-ras mutated T3-4 and/or N+, M0 disease by MRI were included. Recommended doses from phase I part consisted of RT 1.8 Gy/day x25 with Cape 825mg/m2bid x 33 in combination with Sor 400mg/d. The primary endpoint for the phase II part was pathological complete response (pCR) prospectively defined as grade 3 (near complete regression) or 4 (complete regression) in the histological grading system according to Dworak (DC). A pCR rate of 8% or lower was considered uninteresting and of 22% or higher was promising. Secondary endpoints included sphincter preservation, R0 resection, downstaging and safety. Results: 54 pts were treated in 18 centers in Switzerland und Hungary, 40 pts were included into the single arm phase II part. Median dose intensity per day was 100.0% for RT, 98.6% for Cape and 100.0% for Sor respectively. pCR rate was 60.0% (95%CI: 43.3%, 75.1%) by central independent pathological review (15.0% DC grade 4; 45.0% DC grade 3). Sphincter preservation was achieved in 89.5%, R0 resection in 94.7% and downstaging in 81.6% of the pts. The most common grade 3 toxicities included diarrhea (15.0%), skin toxicity outside of the RT field (12.5%), pain (7.5%), skin toxicity in RT field, proctitis, fatigue and cardiac ischemia (each 5.0%). Laboratory AEs grade 3/4 were neutropenia (1 pt grade 4; 1 grade 3), creatinine elevation (1 pt grade 3). Conclusions: The combination of Sor to standard RCT with Cape in k-ras mutated LARC tumors is highly active with acceptable toxicity and deserves further investigation.

Fibroblast growth factor 2 restrains ras-driven proliferation of malignant cells by triggering RhoA-mediated senescence

Fibroblast growth factor 2 (FGF2) is considered to be a bona fide oncogenic factor, although results from our group and others call this into question. Here, we report that exogenous recombinant FGF2 irreversibly inhibits proliferation by inducing senescence in Ras-dependent malignant mouse cells, but not in immortalized nontumorigenic cell lines. We report the following findings in K-Ras-dependent malignant YI adrenocortical cells and H-Ras V12-transformed BALB-3T3 fibroblasts: (a) FGF2 inhibits clonal growth and tumor onset in nude and immunocompetent BALB/c mice, (b) FGF2 irreversibly blocks the cell cycle, and (c) FGF2 induces the senescence-associated -galactosidase with no accompanying signs of apoptosis or necrosis. The tyrosine kinase inhibitor PD173074 completely protected malignant cells from FGF2. In Yl adrenal cells, reducing the constitutively high levels of K-Ras-GTP using the dominant-negative RasN17 mutant made cells resistant to FGF2 cytotoxicity. In addition, transfection of the dominant-negative RhoA-N19 into either YI or 3T3-B61 malignant cell lines yielded stable clonal transfectants that were unable to activate RhoA and were resistant to the FGF2 stress response. We conclude that in Rasdependent malignant cells, FGF2 interacts with its cognate receptors to trigger a senescence-like process involving RboAGTP. Surprisingly, attempts to select FGF2-resistant cells from the Yl and 3T3-B61 cell lines yielded only rare clones that (a) had lost the overexpressed ras oncogene, (b) were dependent on FGF2 for proliferation, and (c) were poorly tumorigenic. Thus, FGF2 exerted a strong negative selection that Rasdependent malignant cells could rarely overcome.

Compartmentalization of Ras proteins

The Ras GTPases operate as molecular switches that link extracellular stimuli with a diverse range of biological outcomes. Although many studies have concentrated on the protein-protein interactions within the complex signaling cascades regulated by Ras, it is becoming clear that the spatial orientation of different Ras isoforms within the plasma membrane is also critical for their function. H-Ras, N-Ras and K-Ras use different membrane anchors to attach to the plasma membrane. Recently it has been shown that these anchors also act as trafficking signals that direct palmitoylated H-Ras and N-Ras through the exocytic pathway to the cell surface but divert polybasic K-Ras around the Golgi to the plasma membrane via an as yet-unidentified-route. Once at the plasma membrane, H-Ras and :K-Ras operate in different microdomains. K-Ras is localized predominantly to the disordered plasma membrane, whereas H-Ras exists in a GTP-regulated equilibrium between disordered plasma membrane and cholesterol-rich lipid rafts. These observations provide a likely explanation for the increasing number of biological differences being identified between the otherwise highly homologous Ras isoforms and raise interesting questions about the role membrane microlocalization plays in determining the interactions of Ras with its effecters and exchange factors.

GTP-dependent segregation of H-ras from lipid rafts is required for biological activity

Different sites of plasma membrane attachment may underlie functional differences between isoforms of Ras. Here we show that palmitoylation and farnesylation targets H-ras to lipid rafts and caveolae, but that the interaction of H-ras with these membrane subdomains is dynamic. GTP-loading redistributes H-ras from rafts into bulk plasma membrane by a mechanism that requires the adjacent hypervariable region of H-ras. Release of H-ras-GTP from rafts is necessary for efficient activation of Raf. By contrast, K-ras is located outside rafts irrespective of bound nucleotide. Our studies identify a novel protein determinant that is required for H-ras function, and show that the GTP/GDP state of H-ras determines its lateral segregation on the plasma membrane.

The linker domain of the Ha-Ras hypervariable region regulates interactions with exchange factors, Raf-1 and phosphoinositide 3-kinase

Ha-Ras and Ki-Ras have different distributions across plasma membrane microdomains. The Ras C-terminal anchors are primarily responsible for membrane microlocalization, but recent work has shown that the interaction of Ha-Ras with lipid rafts is modulated by GTP loading via a mechanism that requires the hypervariable region (HVR). We have now identified two regions in the HVR linker domain that regulate Ha-Ras raft association. Release of activated Ha-Ras from lipid rafts is blocked by deleting amino acids 173-179 or 166-172. Alanine replacement of amino acids 173-179 but not 166-172 restores wild type micro-localization, indicating that specific N-terminal sequences of the linker domain operate in concert with a more C-terminal spacer domain to regulate Ha-Ras raft association. Mutations in the linker domain that confine activated Ha-RasG12V to lipid rafts abrogate Raf-1, phosphoinositide 3-kinase, and Akt activation and inhibit PC 12 cell differentiation. N-Myristoylation also prevents the release of activated Ha-Ras from lipid rafts and inhibits Raf-1 activation. These results demonstrate that the correct modulation of Ha-Ras lateral segregation is critical for downstream signaling. Mutations in the linker domain also suppress the dominant negative phenotype of Ha-RasS17N, indicating that HVR sequences are essential for efficient interaction of Ha-Ras with exchange factors in intact cells.

Ras proteins: Different signals from different locations

Ras signalling has classically been thought to occur exclusively at the inner surface of a relatively uniform plasma membrane. Recent studies have shown that Ras proteins interact dynamically with specific microdomains of the plasma membrane as well as with other internal cell membranes. These different membrane microenvironments modulate Ras signal output and highlight the complex interplay between Ras location and function.