The fusogenic peptide HA2 impairs selectivity of CXCR4-targeted protein nanoparticles.

We demonstrate here that the genetic incorporation of the fusogenic peptide HA2 into a CXCR4-targeted protein nanoparticle dramatically reduces the specificity of the interaction between nanoparticles and cell receptors, a factor to be considered when designing tumor-homing drug vehicles displaying endosomal-escape agents. The loss of specificity is concomitant with enhanced cell penetrability.

Gene expression signatures and molecular markers associated with clinical outcome in locally advanced head and neck carcinoma.

The purpose of this study was to identify molecular markers associated with tumor recurrence and survival in patients with locally advanced head and neck squamous cell carcinoma (HNSCC). We studied the expression profile of 63 pre-treatment tumor biopsies obtained from locally advanced HNSCCs treated with standard treatments. Cluster analysis identified three tumor subtypes associated with significant differences in local recurrence-free survival (LRFS) (P<0.001), progression free-survival (PFS) (P<0.009) and overall survival (OS) (P<0.004). Tumor subtype 1, associated with short LRFS, PFS and OS, showed features of epithelial-mesenchymal transition and undifferentiation. It also overexpressed genes involved in cell adhesion, NF-κB and integrin signalling. Tumor subtype 3, associated with longer LRFS, PFS and OS, showed a high degree of differentiation and overexpressed genes located in chromosomal regions 19q13 and 1q21. Tumor subtype 2, which had an intermediate clinical outcome between subtype 1 and subtype 3, overexpressed genes involved in branching morphogenesis. Finally, we validated the association between gene cluster classification and patient survival using Gene Set Enrichment Analysis and two HNSCC data sets obtained from two independent patient cohorts. In conclusion, we generated a gene prognostic signature associated with survival in locally advanced patients using the expression profile of the pre-treatment tumor biopsy. Independent prospective studies would be necessary to assess if the proposed survival signature could help to guide clinical management of HNSCC.

Mouse models in oncogenesis and cancer therapy.

Animal models have been critical in the study of the molecular mechanisms of cancer and in the development of new antitumor agents; nevertheless, there is still much room for improvement. The relevance of each particular model depends on how close it replicates the histology, physiological effects, biochemical pathways and metastatic pattern observed in the same human tumor type. Metastases are especially important because they are the main determinants of the clinical course of the disease and patient survival, and are the target of systemic therapy. The generation of clinically relevant models using the mouse requires their humanization, since differences exist in transformation and oncogenesis between human and mouse. Although genetically modified (GM) mice have been instrumental in understanding the molecular mechanisms involved in tumor initiation, they have been less successful in replicating advanced cancer. Moreover, a particular genetic alteration frequently leads to different tumor types in human and mouse and to lower metastastatic rates in GM mice than in humans. These findings question the capacity of current GM mouse carcinoma models to predict clinical response to therapy. On the other hand, orthotopic (ORT) xenografts of human tumors, or tumor cell lines, in nude mice reproduce the histology and metastatic pattern of most human tumors at advanced stage. Using ex vivo genetic manipulation of human tumor cells, ORT models can be used to molecularly dissect the metastatic process and to evaluate in vivo tumor response to therapy, using non-invasive procedures. Nevertheless, this approach is not useful in the study of the initial stages of tumorigenesis or the contribution of the immune system in this process. Despite ORT models are more promising than the most commonly used subcutaneous xenografts in preclinical drug development, their capacity to predict clinical response to antitumor agents remains to be studied. Humanizing mouse models of cancer will most likely require the combined use of currently available methodologies.

K-ras and p16 aberrations confer poor prognosis in human colorectal cancer.

PURPOSE: Mutations in the K-ras gene are frequent in human cancer. ras activation in primary cells results in a cellular senescence phenotype that is precluded by inactivation of p16. At the clinical level, this may imply a differential behavior for tumors with alternative or cooperative activation of K-ras function and impairment of p16 pathways. PATIENTS AND METHODS: We have determined the presence of mutations in the K-ras gene and the methylation status of p16 promoter in a series of 119 prospectively collected colorectal carcinomas. p53 mutations and p14 alternative reading frame methylation status were also assessed. Associations with survival were investigated. RESULTS: K-ras mutations were present in 44 (38%) of 115 cases, and p16 methylation was present in 42 (37%) of 113 cases. p53 mutations were detected in 50% (56 of 115) and p14 methylation in 29% (32 of 112) of cases. K-ras and p16 alterations were independent genetic events. Presence of K-ras or p16 genetic alterations (analyzed independently) was associated with shorter survival, although differences were not statistically significant. Cox analysis of the two variables combined showed a diminished survival as the results of an interaction between p16 and K-ras. Alternative alteration of K-ras and p16 genes was an independent prognostic factor in human colorectal cancer in univariate and multivariate analysis. Differences were maintained when cases undergoing radical surgery and without distant metastases were considered. CONCLUSION: These results suggest that the combined K-ras and p16 analyses may be of prognostic use in human colorectal cancer.

K-ras codon 12 mutation induces higher level of resistance to apoptosis and predisposition to anchorage-independent growth than codon 13 mutation or proto-oncogene overexpression.

The position of the point mutation in the c-K-ras gene appears associated with different degrees of aggressiveness in human colorectal tumors. In addition, colon tumors carrying K-ras codon 12 mutations associate with lower levels of apoptosis than tumors lacking this mutation. To test the hypothesis of a distinct transforming capacity of different K-ras forms in an in vitro system, we generated stable transfectants of NIH3T3 cells expressing a plasmid containing K-ras mutated at codon 12 (K12) or at codon 13 (K13), or overexpressing the K-ras proto-oncogene (Kwt-oe). We evaluated changes in morphology, proliferative capacity, contact inhibition, and predisposition to apoptosis and anchorage-independent growth in K12, K13, and Kwt-oe transformants. In addition, we studied alterations in expression and/or activation of proteins that participate in signal transduction downstream of Ras or are involved in the regulation of apoptosis and cell-cell (E-cadherin and beta-catenin) and cell-substrate (focal adhesion kinase) interactions. We observed that K13 or Kwt-oe transformants died synchronically 24-48 h after reaching confluency. Their death was apoptotic. In contrast, K12 grew, forming bigger colonies with higher cell densities; and before reaching confluency, spontaneously formed spheroids and showed no sign of apoptosis. The enhanced resistance to apoptosis, loss of contact inhibition, and predisposition to anchorage-independent growth in the K12 transformants were associated with higher AKT/protein kinase B activation, bcl-2, E-cadherin, beta-catenin, and focal adhesion kinase overexpression, and RhoA underexpression, whereas the increased sensitivity of K13 or Kwt-oe transformants to apoptosis was associated with increased activation of the c-Jun-NH2-terminal kinase 1 pathway. All transformants showed a similar overactivation of mitogen-activated protein kinases and levels of bax expression similar to the endogenous level. Therefore, in our in vitro model, the localization of the mutation in the K-ras gene predisposes to a different level of aggressiveness in the transforming phenotype. K12 may increase aggressiveness not by altering proliferative pathways, but by the differential regulation of K-Ras downstream pathways that lead to inhibition of apoptosis, enhanced loss of contact inhibition, and increased predisposition to anchorage-independent growth. These results offer a molecular explanation for the increased aggressiveness of the tumors with K-ras codon 12 mutations observed in the clinical setting.

NF1 inactivation cooperates with N-ras in in vivo lymphogenesis activating Erk by a mechanism independent of its Ras-GTPase accelerating activity.

We crossed transgenic mice overexpressing the N-ras proto-oncogene (RasTg) with mice carrying one inactivated copy of the NF1 tumor suppressor gene (NF1+/-) to assess their possible cooperation in tumorigenesis. We have found a significant increase in the incidence of lymphomas in animals with both lesions (RasTg NF1+/-), as compared with animals with single lesions. The mechanism of this cooperation appears to be independent of the NF1 GTPase activating activity since the level of Ras-GTP in primary cultures of tumor tissue do not differ among animals with double and with single lesions. Nevertheless, the finding of significantly higher levels of Erk-1 and Erk-2 activation in lymphomas in the RasTg NF1+/- than in the RasTg group suggests that this cooperative effect may be in part explained by increased signaling through the Erk pathways. Consistent with a role for Erk activation in transformation is the additional observation that Erk-1 and Erk-2 activation is significantly increased in lymphomas as compared with normal spleen. This activation is likely to occur by phosphorylation of previously synthesized and inactive Erk proteins since, despite differences in activation, Erk-1 and Erk-2 expression is similar in normal and lymphoid tissue in all groups. The observed cooperation in in vivo lymphomagenesis between N-ras overexpression and NF1 inactivation emphasizes the importance of searching for additional functions for the NF1 protein and of intensifying the screening for NF1 mutations in human lymphomas.

Antitumor effect of a farnesyl protein transferase inhibitor in mammary and lymphoid tumors overexpressing N-ras in transgenic mice.

We tested the antineoplastic effect of the farnesyltransferase inhibitor L-744,832 in mammary and lymphoid tumors overexpressing the N-ras proto-oncogene in transgenic mice. Mice bearing mammary tumors were randomly assigned to receive daily 40 mg/kg s.c. injections of this compound (experimental group, n = 6) or vehicle (control group, n = 6) per day for 5.5 weeks. Treatment with the compound significantly reduced the mammary tumor mean growth rate in the experimental group (-0.7 mm3/day), as compared with the control group (+28.2 mm3/day; P < 0.001). There was a significant difference in lymphoma incidence at the end of the treatment between the experimental (0 of 6) and the control (3 of 6) groups (P < 0.05). Therefore, this compound is effective in treating in vivo mammary carcinomas and lymphomas in which an activated N-Ras pathway drives tumorigenesis. The number of apoptotic figures in mammary tumors was significantly higher (P = 0.04) in the experimental (14.7 +/- 8.1) than it was in the control (5.7 +/- 3.5) group, indicating that apoptotic induction could contribute to the mechanism of antitumor activity of this compound. We analyzed the level of processing of N-Ras and H-Ras after immunoprecipitation and Western blotting of protein extracts obtained from mammary tumors treated with L-744,832 or vehicle, either in vivo or in vitro (after primary culture of the same tumors), and from several in vitro treated control cell lines. In all compound-treated mammary tumors and cell lines, H-Ras was mostly unprocessed (more so after in vitro than after in vivo treatment), whereas N-Ras remained mostly processed. Both H-Ras and N-Ras remained fully processed in all vehicle-treated samples. These findings are consistent with a less intense antineoplastic effect of the treatment with the compound in our N-ras model than the effect previously reported for the same compound in H-ras transgenics. In addition, the finding that, in compound-treated mammary tumors, the N-Ras protein remains mainly processed suggests that, in our model, other proteins in addition to Ras may be a target for the compound. Our results and the previous findings of frequent N-ras activation in human hematopoietic malignancies support a role for L-744,832 in the treatment of lymphomas and of mammary carcinomas with an activated N-Ras pathway, as well as the testing of a farnesyl protein transferase inhibitor in humans to establish its clinical relevance.

Isolation of high molecular weight DNA for reliable genotyping of transgenic mice.

A fast and reliable method for the PCR characterization of DNA from mouse toes is described. The toes biopsied to tag the mice are incubated for 2 h in proteinase K and heated for 15 min at 95 degrees C. This DNA solution is directly used as a template for PCR amplification. The same procedure can be used for PCR analysis of DNA from other tissues in adult mice, mouse embryos and cultured cells. Because of minimal tissue manipulation, high-quality and high-molecular-weight DNA (fragments larger than 100-200 kb) is isolated. This procedure is performed in a single tube and requires no organic solvent extraction or centrifugation, allowing the isolation of high-molecular-weight DNA suitable for PCR amplification in a fast and reproducible way. Only the tissue excised during mice tagging is used and a large number of animals can be quickly and simultaneously analyzed as required to maintain a transgenic mice colony. In addition, this rapid and efficient procedure represents an alternative to other methods in which, in our experience, inhibition of the PCR amplification occurs when DNA from tail tissues is used.

Inactivation of the cyclin-dependent kinase inhibitor p15INK4b by deletion and de novo methylation with independence of p16INK4a alterations in murine primary T-cell lymphomas.

A wide panel of murine induced T-cell lymphomas have been analysed for p16INK4a or p15INK4b alterations. Only one gamma-radiation-induced lymphoma showed p16INK4a homozygous deletion and no other intragenic mutations were found in these INK4 genes. However, de novo methylation of the 5' CpG islands of the murine p15INK4b and p16INK4a genes was found to be highly frequent. While p16INK4a hypermethylation was found in 36% of the neutron-radiation-induced lymphomas and 15% of the gamma-radiation-induced lymphomas, de novo methylation of p15INK4b occurs in 88% and 42% of these tumors respectively, correlating with deficient expression of the corresponding mRNA and allelic losses in the p15INK4b and p16INK4a chromosome location. These data represent, to our knowledge, the first report on the significant involvement of hypermethylation of these INK4 genes in murine primary tumors. Moreover, they show the importance of allelic losses and CpG island methylation of p15INK4b gene inactivation and support a tumor suppressor role for p15INK4b in T-cell lymphomas independent of p16INK4a.

S49 cells endogenously express subtype 2 somatostatin receptors which couple to increase protein tyrosine phosphatase activity in membranes and down-regulate Raf-1 activity in situ.

S49 cells expressed type 2 somatostatin receptors (sstr2) by immunoblotting. Analysis by reverse transcription and polymerase chain reaction (RT-PCR) methodologies showed that S49 cells express predominantly sstr2A and sstr2B mRNAs; other subtypes were either not detected, in the case of sstr1, sstr3, sstr4, or variably detected, in the case of sstr5. No mutations were present in S49 cells at codon 12, 13, or 61 of the N-, K-, or H-ras genes. Nevertheless, randomly growing S49 cells contained Raf-1 activity by specific immune complex kinase assays. Treatment of S49 cells with somatostatin transiently inactivated the basal activity of Raf-1, but not that of B-Raf. Addition of somatostatin plus guanyl-5'-yl imidodiphosphate (GMPPNP) to S49 membranes stimulated PTPase activity. The concentration dependence for stimulation of PTPase activity correlated with high affinity binding of [125I-Tyr11]somatostatin-14. Both the effect of somatostatin to stimulate PTPase activity and to inactivate Raf-1 were abrogated by PTx. PTPase activity stimulated by somatostatin plus GMPPNP was recovered in a peak of high apparent M(r) (670,000) after solubilisation with Triton X-100 and Superose 6 chromatography. Furthermore, addition of activated, brain G alpha i/o subunits to fractions from control membranes stimulated PTPase activity in the high M(r) peak. Thus, S49 membranes contain a G-protein regulated PTPase (PTPase-G), and PTPase-G in these cells may reside in a high molecular weight complex.

Activated N-ras oncogene and N-ras proto-oncogene act through the same pathway for in vivo tumorigenesis.

We compared the tumorigenic effects of the N-ras oncogene and the N-ras proto-oncogene in lymphoid and mammary tissues in an in vivo model. For this purpose, we generated transgenic mice with high levels of N-ras oncogene or N-ras proto-oncogene expression, driven by the complete mouse mammary tumor virus LTR (MMTV-LTR) (MMTV/N-rasT and MMTV/N-rasN constructs) and transgenic mice with low levels of N-ras oncogene or N-ras proto-oncogene expression, driven by a truncated MMTV-LTR (TMTV/N-rasT and TMTV/N-rasN constructs). We show that both, the N-ras proto-oncogene and the N-ras oncogene with a C:G-->A:T mutation at codon 61, lead to identical tumor types: lymphoblastic T-cell lymphomas, cleaved B-cell lymphomas and poorly differentiated mammary carcinomas. Nevertheless, there were quantitative differences in tumor incidence and latency and in transgene expression among N-ras oncogene and N-ras proto-oncogene transgenics. Despite these differences in tumor kinetics, the predisposition to identical tumor types is in agreement with the idea that the N-ras oncogene and the N-ras proto-oncogene act through the same pathway for in vivo tumorigenesis in B-cells, T-cells or mammary epithelial cells.

Promoter demethylation in MMTV/N-rasN transgenic mice required for transgene expression and tumorigenesis.

We studied demethylation within the transgene promoter in transgenic mice carrying the N-ras proto-oncogene driven by the mouse mammary tumor long terminal repeat (MMTV/N-rasN) and the relationship of demethylation to transgene overexpression and tumorigenesis. Demethylation at Fspl or Clal sites correlated with age of the animal and transgene expression in nontumorous mammary gland. Demethylation preceded expression in this tissue. In lymphomas and mammary tumors, the promoter Fspl and Clal sites were significantly more demethylated than in nontumorous control tissues. The Aval, Cfol, and Hpall sites were also found to be undermethylated in older animals and showed differences between tumor and control tissues. Two additional sites (Eagl and Narl) remained fully methylated in all tissues. In contrast with normal tissue, demethylation at the Fspl and Clal sites and expression were not correlated in tumor tissue. An increase in expression in normal tissue initially occurred and was correlated with the level of promoter demethylation; this increase was followed by a further increment in transgene expression when tumors developed. Thus, promoter demethylation leading to transgene overexpression was associated with long-latency tumorigenesis in MMTV/N-rasN transgenic mice. Demethylation of proto-oncogene promoters may therefore be a mechanism of carcinogenesis that requires further investigation in human tumors.

An overexpressed N-ras proto-oncogene cooperates with N-methylnitrosourea in mouse mammary carcinogenesis.

The induction of tumors with chemicals and the production of transgenic animals are two experimental approaches to study oncogene involvement in carcinogenesis. The combination of both strategies offers an excellent model system to study tumor development. This study analyzes the potential cooperation of N-methylnitrosourea (MNU) treatment and N-ras proto-oncogene overexpression in tumorigenesis in transgenic mice. The overexpression of the N-ras proto-oncogene in these animals is associated with development of mammary tumors and lymphomas. After MNU treatment we analyzed tumor incidence and latency, levels of transgene expression, and pattern of ras mutations in codons 12, 13, and 61 of H-, K-, and N-ras genes in both tumor types. Transgenic mice treated with MNU had significantly (P < 0.001) shorter latency of appearance of mammary tumors [8.6 +/- 3.0 (SD) months] than phosphate-buffered saline-treated transgenics (12.8 +/- 2.3 months). All mammary tumors overexpressed the N-ras transgene and lacked ras mutations. Moreover, MNU-treated transgenics had an incidence and latency of lymphomas similar to that of MNU-treated nontransgenic mice. No significant differences in incidence of point mutations (K-ras codon 12 or 13 and N-ras codon 61) in lymphomas were seen between these two groups. All lymphomas overexpressed the N-ras transgene, except for those carrying a K-ras point mutation. Overexpression of the N-ras proto-oncogene cooperates with non-ras genes mutated by MNU in mouse mammary carcinogenesis. Conversely, N-ras proto-oncogene overexpression does not show cooperation with MNU in lymphomagenesis in our system. This study suggests that proto-oncogene overexpression may be a mechanism of activation of the ras pathway, alternative to point mutation. Similarly to actions for ras genes activated by point mutation, overexpression of the N-ras protooncogene predisposes to tumorigenesis and cooperates with a carcinogen in tumorigenesis. The possibility that ras overexpression plays a role in human breast tumorigenesis requires active investigation.

Brain interleukin-1 beta in Alzheimer's disease and vascular dementia.

Recent investigations indicate that a neuroimmune reaction, associated with inflammatory mechanisms, can contribute in Alzheimer's disease (AD) to cell damage and neurodegeneration. Activation of microglial cells, expression of immunohistochemical markers of brain immune function, the presence of complement proteins in brain tissue and changes in cytokine production have been reported in AD. We have studied the concentration of interleukin-1 beta (IL-1 beta) in different regions of the central nervous system (CNS) in post-mortem samples from patients with AD or vascular dementia (VD) and in age-matched control subjects (CS). IL-1 beta levels were significantly higher in AD than in VD or CS in the frontal cortex, parietal cortex, temporal cortex, hypothalamus, thalamus and hippocampus. The highest increases in IL-1 beta levels were observed in the frontal cortex (CS = 0.75 +/- 0.045; AD = 2.47 +/- 0.12, p < 0.001; VD = 1.52 +/- 0.078 pg/mg, p < 0.001) and hippocampus (CS = 0.71 +/- 0.042; AD = 2.63 +/- 0.19, p < 0.001; VD = 1.21 +/- 0.23 pg/mg, p < 0.01). No significant changes were detected in the occipital cortex and cerebellum in either AD or VD. These results clearly demonstrate that demented patients show a generalized increment of IL-1 beta production in the CNS, with maximum response in those brain regions where AD neuropathology is most prominent. This overall increase in cytokine production might represent an early event in the activation of a neuroimmune cascade leading to cell death and neurodegeneration in brain regions where a primary cause (e.g., genetic, toxic, vascular) facilitates the induction of resting microglia for firing brain immune function.

Absence of MDM-2 gene amplification in experimentally induced tumors regardless of p53 status.

To assess the generality of the hypothesis that murine double-minute-2 (MDM-2) gene amplification complements the absence of p53 mutation during tumor development, we analyzed 143 murine tumors induced by a variety of carcinogenic agents in two different mouse strains. Only three of 143 tumors showed p53 genetic alterations and none showed MDM-2 amplification, indicating the existence of alternative pathways that permit tumor cells to bypass p53-MDM-2 control.

Overexpression of the N-ras proto-oncogene, not somatic mutational activation, associated with malignant tumors in transgenic mice.

We have produced transgenic mice that carry a foreign gene construct consisting of the N-ras proto-oncogene driven by the mouse mammary tumor virus (MMTV) long terminal repeat. Overexpression of the normal N-ras gene is associated with development of hyperplasias and tumors in a variety of tissues. The tumors are clearly malignant, as evidenced by the presence of metastatic lesions. Extensive analysis of the foreign ras gene in these tumors by use of polymerase chain reaction and sequencing demonstrates in all cases the absence of somatically acquired mutations at those codons normally associated with activation of the ras genes. Thus, these tumors develop from overexpression of the proto-oncogene rather than the presence of the mutated oncogene. These data demonstrate that overexpression of a protooncogene of the ras family can predispose cells in vivo to fully malignant behavior.

ras activation in experimental carcinogenesis.

We review experimental models of carcinogenesis in which the role of ras activation has been most thoroughly studied: skin, thymus, mammary gland and liver. Qualitative changes (point mutations), as well as quantitative changes (over-expression, increased gene dosage) contribute to the transforming phenotype induced by ras genes. The activation of the three different ras family members is associated with particular tumor types, carcinogenic agents, and carcinogenic stages, suggesting the ras proteins may be involved in different biological functions. Depending on the system, ras activation has been shown to be an early and/or a late event in the multi-step process of carcinogenesis. These data underscore the possible relationship between ras activation and cell type specificity, proliferation, differentiation or cell-cell interaction.

Tumorigenesis and male sterility in transgenic mice expressing a MMTV/N-ras oncogene.

Transgenic mice carrying the activated N-ras oncogene under the transcriptional control of the mouse mammary tumor virus (MMTV) long terminal repeat were produced. The transgene is expressed in a tissue distribution consistent with the fact that it is driven by the MMTV-LTR, and similarly to MMTV/H-ras constructs, its presence elicits tumors in Harderian, mammary and salivary glands. In addition it appears to compromise male reproductive function, which has not been described with the other ras transgenes. This finding is consistent with the existence of distinct physiological actions for each of the ras family members.