Stromal disrupting effects of nab-paclitaxel in pancreatic cancer.

BACKGROUND: Nab-paclitaxel and gemcitabine have demonstrated a survival benefit over gemcitabine alone in advanced pancreatic cancer (PDA). This study aimed to investigate the clinical, biological, and imaging effects of the regimen in patients with operable PDA. METHODS: Patients with operable PDA received two cycles of nab-paclitaxel and gemcitabine before surgical resection. FDG-PET and CA19.9 tumour marker levels were used to measure clinical activity. Effects on tumour stroma were determined by endoscopic ultrasound (EUS) elastography. The collagen content and architecture as well as density of cancer-associated fibroblasts (CAFs) were determined in the resected surgical specimen and compared with a group of untreated and treated with conventional chemoradiation therapy controls. A co-clinical study in a mouse model of PDA was conducted to differentiate between the effects of nab-paclitaxel and gemcitabine. RESULTS: A total of 16 patients were enrolled. Treatment resulted in significant antitumour effects with 50% of patients achieving a >75% decrease in circulating CA19.9 tumour marker and a response by FDG-PET. There was also a significant decrement in tumour stiffness as measured by EUS elastography. Seven of 12 patients who completed treatment and were operated had major pathological regressions. Analysis of residual tumours showed a marked disorganised collagen with a very low density of CAF, which was not observed in the untreated or conventionally treated control groups. The preclinical co-clinical study showed that these effects were specific of nab-paclitaxel and not gemcitabine. CONCLUSION: These data suggest that nab-paclitaxel and gemcitabine decreases CAF content inducing a marked alteration in cancer stroma that results in tumour softening. This regimen should be studied in patients with operable PDA.

To cycle or not to cycle: a critical decision in cancer.

Tumour cells undergo uncontrolled proliferation, yet tumours most often originate from adult tissues, in which most cells are quiescent. So, the proliferative advantage of tumour cells arises from their ability to bypass quiescence. This can be due to increased mitogenic signalling and/or alterations that lower the threshold required for cell-cycle commitment. Understanding the molecular mechanisms that underlie this commitment should provide important insights into how normal cells become tumorigenic and how new anticancer strategies can be devised.

Loss of Cdk4 expression causes insulin-deficient diabetes and Cdk4 activation results in beta-islet cell hyperplasia.

To ascertain the role of cyclin-dependent kinase 4 (Cdk4) in vivo, we have targeted the mouse Cdk4 locus by homologous recombination to generate two strains of mice, one that lacks Cdk4 expression and one that expresses a Cdk4 molecule with an activating mutation. Embryonic fibroblasts proliferate normally in the absence of Cdk4 but have a delayed S phase on re-entry into the cell cycle. Moreover, mice devoid of Cdk4 are viable, but small in size and infertile. These mice also develop insulin-deficient diabetes due to a reduction in beta-islet pancreatic cells. In contrast, mice expressing a mutant Cdk4 that cannot bind the cell-cycle inhibitor P16INK4a display pancreatic hyperplasia due to abnormal proliferation of beta-islet cells. These results establish Cdk4 as an essential regulator of specific cell types.

Neurotrophic factors and their receptors.

Several new members of the nerve growth factor family of neurotrophins, which comprises nerve growth factor itself, brain-derived neurotrophic factor and neurotrophins-3, -4 (also known as neurotrophin-5) and -6, have been isolated in recent years. Their signaling receptors have been identified as the Trk family of tyrosine protein kinases, thus facilitating the dissection of the signaling pathways responsible for mediating their trophic properties. More recently, the advent of gene targeting has made it possible to generate strains of mice lacking neurotrophins and their receptors. Analysis of the phenotypes of these mutant animals has provided detailed information on the role that neurotrophins and their receptors play in the ontogeny of the mammalian nervous system.

Viral genes involved in leukemogenesis. I. Generation of recombinants between oncogenic and nononcogenic mouse type-C viruses in tissue culture.

An approach toward elucidation of the mechanisms of action of mammalian leukemia viruses has been made by the generation in tissue culture of recombinant viruses between a potent murine leukemia virus (MuLV), Rauscher-MuLV, and an endogenous xenotropic mouse type-C virus, BALB:virus-2, without known malignant potential. Using a double selection system devised to select against the temperature-sensitive (ts) lesion associated with a mutant of Rauscher-MuLV and the xenotropic host range of BALB:virus-2, recombinant viruses were obtained at frequencies ranging from 0.01 to 0.1%. Recombinant viruses were identified on the basis of the type specific antigenic determinants in the translational products of gag (p15, p12, p30, and p10 proteins), pol (reverse transcriptase), and env (gp70 glycoprotein) genes. By this approach, the partial genetic maps of a large number of recombinants were obtained. The fact that p10 of Rauscher-MuLV ts 25, the mutant utilized, was the only protein uniformly lacking in recombinant viruses, localized the lesion inhibiting gag precursor cleavage in this mutant at the carboxy terminus of its gag gene. The recombinant viruses demonstrated two host range phenotypes as defined by Fv-1 host cell restriction. In each case, NB-tropic recombinants possessed the p30 of BALB:virus-2 p30. Thus, it was possible to assign the site of Fv-1 action at, or closely linked, to the viral p30. The target within the viral genome of a second host restriction was also mapped. A serum factor, previously shown to specifically inactivate xenotropic virus infectivity, was demonstrated to exert its action on the viral env gene product. The system described here allows the generation of specific recombinant genotypes that should be useful in defining those regions of the viral genome involved in leukemogenesis.

Wild mouse RNA tumor viruses. A nongenetically transmitted virus group closely related to exogenous leukemia viruses of laboratory mouse strains.

Type-C RNA viruses isolated from wild mice are causative of naturally occurring neoplasia and neurologic diseases. Biochemical and immunologic characterization of this virus group revealed that amphotropic viruses isolated from wild mice trapped in separate geographical areas are indistinguishable, whereas amphotropic and ecotropic viruses naturally infecting the same animal are env gene variants. Molecular hybridization studies established that neither host range variant is endogenous to the Mus musculus genome, although each demonstrates partial nucleotide sequence homology. Wild mouse type-C viruses exhibited much closer molecular and antigenic relatedness to the exogenous virus subgroup (Friend-, Moloney-, and Rauscher-MuLV) than to prototype endogenous viruses isolated from laboratory mouse strains. The evidence indicates that exogenous mouse type-C viruses have been maintained in nature over a long period of evolution as a separate virus group, causative of tumors in mice by a mechanism solely involving their transmission as infectious agents.

Tyrosine phosphorylation of the vav proto-oncogene product in activated B cells.

Activation of B lymphocytes by engagement of their immunoglobulin M antigen receptors results in phosphorylation of a number of proteins on tyrosine residues. One such protein is p95vav, the product of the vav proto-oncogene. Tyrosine phosphorylation of p95vav occurred within seconds of immunoglobulin M cross-linking and was independent of other events induced during stimulation of B cells, such as protein kinase C activation, guanosine triphosphate-binding protein signaling, and calcium mobilization. Moreover, engagement of antigen receptors induced the rapid (approximately 5 seconds) and transient (approximately 60 seconds) association of p95vav with a 70-kilodalton tyrosine-phosphorylated protein, Vap-1, an interaction mediated by the Src homology 2 domain of p95vav. These results suggest that the vav proto-oncogene participates in the signaling processes that mediate the antigen-induced activation of B lymphocytes.

Independent molecular pathways in initiation and loss of hormone responsiveness of breast carcinomas.

These studies were set up to determine whether those oncogenes participating in the initiation of mammary carcinogenesis (for example, ras oncogenes) play a direct role in the outcome of events associated with the late stages of tumor development such as loss of hormone dependency. Mammary carcinomas induced by a single carcinogenic insult in pubescent rats was selected as an in vivo model system with direct relevance to human breast cancer. Acquisition of hormone-independent growth in these carcinogen-induced tumors was found to be independent of the activation of ras oncogenes during the early stages of carcinogenesis. In agreement with these observations, introduction of a human ras oncogene into human MCF-7 breast carcinoma cells did not abrogate their hormonal dependency for growth in vivo. These findings suggest that those events responsible for the critical stages of breast cancer development occur independently and in an uncoordinated manner.

Activation of ras oncogenes preceding the onset of neoplasia.

The identification of ras oncogenes in human and animal cancers including precancerous lesions indicates that these genes participate in the early stages of neoplastic development. Yet, these observations do not define the timing of ras oncogene activation in the multistep process of carcinogenesis. To ascertain the timing of ras oncogene activation, an animal model system was devised that involves the induction of mammary carcinomas in rats exposed at birth to the carcinogen nitrosomethylurea. High-resolution restriction fragment length polymorphism analysis of polymerase chain reaction-amplified ras sequences revealed the presence of both H-ras and K-ras oncogenes in normal mammary glands 2 weeks after carcinogen treatment and at least 2 months before the onset of neoplasia. These ras oncogenes can remain latent within the mammary gland until exposure to estrogens, demonstrating that activation of ras oncogenes can precede the onset of neoplasia and suggesting that normal physiological proliferative processes such as estrogen-induced mammary gland development may lead to neoplasia if the targeted cells harbor latent ras oncogenes.

Evolutionary relatedness of viper and primate endogenous retroviruses.

A retrovirus previously isolated from a tumored Russell's viper is shown by molecular hybridization to be an endogenous virus of this reptilian species. Radio-immunologic techniques revealed that the viper retrovirus is immunologically and, hence, evolutionarily related to endogenous type D retorviruses of Old World primates. These findings extend the number of vertebrate classes possessing endogenous retroviruses and suggest that type D retroviruses may even be more widely distributed in nature than type C retroviruses.

Malignant activation of a K-ras oncogene in lung carcinoma but not in normal tissue of the same patient.

A single genetic alteration, a guanine-to-cytosine transversion, is responsible for the acquisition of malignant properties by K-ras genes of two human tumor cell lines established from carcinomas of the bladder (A1698) and lung (A2182). As a consequence, arginine instead of the normal glycine is incorporated into the K-ras-coded p21 proteins at amino acid position 12. This mutation creates a restriction enzyme polymorphism that can be used to screen human cells for transforming K-ras genes. This approach was used to identify the mutational event responsible for the malignant activation of a K-ras oncogene in a squamous cell lung carcinoma of a 66-year-old man; this point mutation was not present in either the normal bronchial or parenchymal tissue or in the blood lymphocytes. Hence, malignant activation of a ras oncogene appears to be specifically associated with the development of a human neoplasm.

A transforming ras gene in tumorigenic guinea pig cell lines initiated by diverse chemical carcinogens.

Fetal guinea pig cells were transformed by treatment with four different chemical carcinogens including nitroso compounds and polycyclic hydrocarbons. As a consequence of this treatment, oncogenes capable of transforming NIH/3T3 cells became activated in each of five independently established clonal guinea pig cell lines. Molecular characterization of representative NIH/3T3 transformants revealed that the same oncogene was present in each of the cell lines tested. Moreover, detection of this transforming gene paralleled the acquisition of tumorigenic properties by these neoplastic cells.

Nerve growth factor mediates signal transduction through trk homodimer receptors.

We have investigated the molecular nature of the high affinity nerve growth factor (NGF) receptors by using cell lines expressing gp75LNGFR and gp140trk. Our results suggest that gp75LNGFR and gp140trk interact with NGF independently and that only gp140trk mediates NGF signaling. NGF binds to gp140trk with picomolar affinity and induces its phosphorylation on tyrosine residues regardless of the presence of gp75LNGFR. NGF-gp140trk complexes display the slow dissociation rate and rapid internalization characteristics of high affinity NGF receptors. Cross-linking studies reveal the existence of gp75LNGFR and gp140trk homodimers. However, we were unable to detect gp75LNGFR-gp140trk heterodimers. Coexpression in COS cells of wild-type and kinase deficient mutants reveals that gp140trk receptors can undergo intermolecular phosphorylation, indicating the formation of functional homodimers. Moreover, these kinase deficient mutants inhibit NGF-induced signaling through wild-type gp140trk receptors. These results indicate that the functional high affinity NGF receptors consist of gp140trk homodimeric (or oligomeric) complexes.

The trkB tyrosine protein kinase is a receptor for neurotrophin-4.

Neurotrophin-4 is a novel member of the nerve growth factor family of neurotrophins recently isolated from Xenopus and viper DNA. We now report that the Xenopus NT-4 protein (XNT-4) can mediate some of its biological properties through gp145trkB, a murine tyrosine protein kinase previously identified as a primary receptor for the related brain-derived neurotrophic factor (BDNF). XNT-4 displaces 125I-labeled BDNF from binding to cells expressing gp145trkB receptors, induces their rapid phosphorylation on tyrosine residues, and causes the morphologic transformation of NIH 3T3 cells when coexpressed with gp145trkB. Moreover, XNT-4 induces the differentiation of PC12 cells into sympathetic-like neurons only if they ectopically express gp145trkB receptors. None of these biochemical or biological effects could be observed when XNT-4 was added to cells expressing the related receptors. Replacement of one of the extracellular cysteines (Cys-345) of gp145trkB by a serine residue prevents its activation by XNT-4 but not by BDNF. Therefore, XNT-4 and BDNF may interact with at least partially distinct domains within the gp145trkB receptor.

Ephrin-A5 (AL-1/RAGS) is essential for proper retinal axon guidance and topographic mapping in the mammalian visual system.

Ephrin-A5 (AL-1/RAGS), a ligand for Eph receptor tyrosine kinases, repels retinal axons in vitro and has a graded expression in the superior colliculus (SC), the major midbrain target of retinal ganglion cells. These properties implicate ephrin-A5 in the formation of topographic maps, a fundamental organizational feature of the nervous system. To test this hypothesis, we generated mice lacking ephrin-A5. The majority of ephrin-A5-/- mice develop to adulthood, are morphologically intact, and have normal anterior-posterior patterning of the midbrain. However, within the SC, retinal axons establish and maintain dense arborizations at topographically incorrect sites that correlate with locations of low expression of the related ligand ephrin-A2. In addition, retinal axons transiently overshoot the SC and extend aberrantly into the inferior colliculus (IC). This defect is consistent with the high level of ephrin-A5 expression in the IC and the finding that retinal axon growth on membranes from wild-type IC is inhibited relative to that on membranes from ephrin-A5-/- IC. These findings show that ephrin-A5 is required for the proper guidance and mapping of retinal axons in the mammalian midbrain.

Genetic analysis of ephrin-A2 and ephrin-A5 shows their requirement in multiple aspects of retinocollicular mapping.

Ephrin-A2 and -A5 are thought to be anteroposterior mapping labels for the retinotectal/retinocollicular projection. Here, gene disruptions of both these ephrins are characterized. Focal retinal labeling reveals moderate map abnormalities when either gene is disrupted. Double heterozygotes also have a phenotype, showing an influence of absolute levels. In vitro assays indicate these ephrins are required for repellent activity in the target and also normal responsiveness in the retina. In double homozygotes, anteroposterior order is almost though not completely lost. Temporal or nasal retinal labelings reveal quantitatively similar but opposite shifts, with multiple terminations scattered widely over the target. These results indicate an axon competition mechanism for mapping, with a critical role for ephrins as anteroposterior topographic labels. Dorsoventral topography is also impaired, showing these ephrins are required in mapping both axes.

Topographic guidance labels in a sensory projection to the forebrain.

Visual connections to the mammalian forebrain are known to be patterned by neural activity, but it remains unknown whether the map topography of such higher sensory projections depends on axon guidance labels. Here, we show complementary expression and binding for the receptor EphA5 in mouse retina and its ligands ephrin-A2 and ephrin-A5 in multiple retinal targets, including the major forebrain target, the dorsal lateral geniculate nucleus (dLGN). These ligands can act in vitro as topographically specific repellents for mammalian retinal axons and are necessary for normal dLGN mapping in vivo. The results suggest a general and economic modular mechanism for brain mapping whereby a projecting field is mapped onto multiple targets by repeated use of the same labels. They also indicate the nature of a coordinate system for the mapping of sensory connections to the forebrain.

Similarities and differences in the way neurotrophins interact with the Trk receptors in neuronal and nonneuronal cells.

We have exploited a battery of approaches to address several controversies that have accompanied the expansion of the nerve growth factor (NGF) family of neurotrophic factors and the identification of the Trk tyrosine kinases as receptors for these factors. For example, we find that a recently cloned mammalian neurotrophin, known as either neurotrophin-4 or neurotrophin-5 and assigned widely differing receptor specificities, represents the functional counterpart of Xenopus neurotrophin-4 and is a "preferred" ligand for TrkB. However, its interactions with TrkB can be distinguished from those of brain-derived neurotrophic factor (BDNF) with TrkB. We also find that all of the Trks display similar dose responses to their "preferred" ligands in neuronal as compared with nonneuronal cells (i.e., NGF for TrkA, BDNF and NT-4/5 for TrkB, and NT-3 for TrkC), providing evidence against a role for accessory molecules expressed in neurons in generating receptors that would allow for responses to lower concentrations of the neurotrophins. However, we find that a neuronal environment does restrict the Trks in their ability to respond to their "nonpreferred" neurotrophin ligands.