In vivo oncogenic conflict triggered by co-existing KRAS and EGFR activating mutations in lung adenocarcinoma.

Activating mutations in KRAS and EGFR, the two most frequent oncogenes in human lung adenocarcinoma, are mutually exclusive, a phenotype attributed to functional redundancy implying lack of positive selection. Employing a mouse model expressing EGFRL858R in advanced KrasG12V-driven tumors we show that their mutual exclusivity can be explained by detrimental effects of their co-expression in lung adenocarcinoma. In vivo, expression of EGFRL858R in KrasG12V-driven tumors triggers replicative stress and apoptosis, while the surviving cells enter a transient cytostatic state incompatible with tumor development that is fully reversible upon discontinued EGFRL858R expression. Eventually, sustained expression of both mutants induces attenuation of oncogenic signaling to levels compatible with proliferation and tumor growth resulting in high sensitivity to Mek inhibition. Our results indicate that the mutual exclusivity of KRAS and EGFR mutations occurs as a combination of cellular toxicity and signal adjustment resulting in lack of selective advantage for cells expressing both oncogenes.

Ras signaling is essential for skin development.

Proliferation in the epidermis is a tightly controlled process. During skin development, epidermis formation and hair follicle morphogenesis crucially depend on the regulated balance between proliferation and differentiation. Here we deleted all three Ras loci (H-Ras, N-Ras and K-Ras) from keratinocytes in vitro as well as specifically from the epidermis in mice using a K5Cre strain. Upon Ras elimination, keratinocytes ceased proliferation and entered into senescence without any signs of apoptosis induction. Constitutive activation of the mitogen-activated protein kinase pathway was able to partially rescue the proliferative defects. In mice, Ras signaling was essential for proper development of the epidermis and hair follicles. Deletion of the three Ras loci during epidermis formation in mouse embryos caused a dramatic decrease in proliferation, resulting in a substantially thinner epidermis and delayed appearance of differentiation markers. We could not detect apoptotic or senescent cells in these embryos suggesting that loss of Ras protein expression only leads to severe hypoproliferation. These observations provide genetic evidence for an essential role of Ras proteins in the control of keratinocyte and epidermal proliferation.

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.

Upregulation of miR-21 by Ras in vivo and its role in tumor growth.

miR-21 is a microRNA (miRNA) frequently overexpressed in human cancers. Here we show that miR-21 is upregulated both in vitro and in vivo by oncogenic Ras, thus linking this miRNA to one of the most frequently activated oncogenes in human cancers. Ras regulation of miR-21 occurs with a delayed kinetic and requires at least two Ras downstream pathways. A screen of human thyroid cancers and non-small-cell lung cancers for the expression of miR-21 reveals that it is overexpressed mainly in anaplastic thyroid carcinomas, the most aggressive form of thyroid cancer, whereas in lung its overexpression appears to be inversely correlated with tumor progression. We also show that a LNA directed against miR-21 slows down tumor growth in mice. Consistently, a search for mRNAs downregulated by miR-21 shows an enrichment for mRNAs encoding cell cycle checkpoints regulators, suggesting an important role for miR-21 in oncogenic Ras-induced cell proliferation.

Cyclin-dependent kinase 2 is dispensable for normal centrosome duplication but required for oncogene-induced centrosome overduplication.

Cyclin-dependent kinase 2 (CDK2) has been proposed to function as a master regulator of centrosome duplication. Using mouse embryonic fibroblasts (MEFs) in which Cdk2 has been genetically deleted, we show here that CDK2 is not required for normal centrosome duplication, maturation and bipolar mitotic spindle formation. In contrast, Cdk2 deficiency completely abrogates aberrant centrosome duplication induced by a viral oncogene. Mechanistically, centrosome overduplication in MEFs wild-type for Cdk2 involves the formation of supernumerary immature centrosomes. These results indicate that normal and abnormal centrosome duplication have significantly different requirements for CDK2 activity and point to a role of CDK2 in licensing centrosomes for aberrant duplication. Furthermore, our findings suggest that CDK2 may be a suitable therapeutic target to inhibit centrosome-mediated chromosomal instability in tumor cells.

Cell cycle and cancer: genetic analysis of the role of cyclin-dependent kinases.

Most human tumors harbor mutations that misregulate the early phases of the cell cycle. Here, we summarize genetic evidence, mostly obtained in our laboratory using strains of gene-targeted mice, that provides direct experimental support for a role of Cdk4 in tumor development. Moreover, these genetic studies challenge some well-established concepts regarding the role of Cdks during the early phases of the cell cycle. For instance, they have illustrated that Cdk4 and Cdk6 are not essential for cell division during embryonic development except in the hematopoietic system. More surprisingly, mice lacking Cdk2 survive for over 2 years without detectable abnormalities except in their germ cells, indicating that Cdk2 is essential for meiosis but dispensable for the normal mitotic cell cycle. Cdk2 is also dispensable for cell cycle inhibition and tumor suppression by the Cip/Kip inhibitors, p21(Cip1) and p27(Kip1). These observations have important implications not only to understand cell cycle regulation, but also to validate Cdks as potential targets for the development of therapeutic strategies to block proliferation of tumor cells.

Pancreatic islets from cyclin-dependent kinase 4/R24C (Cdk4) knockin mice have significantly increased beta cell mass and are physiologically functional, indicating that Cdk4 is a potential target for pancreatic beta cell mass regeneration in Type 1 diabetes.

AIMS/HYPOTHESIS: Cyclin-dependent kinase 4 (Cdk4) is crucial for beta cell development. A mutation in the gene encoding for Cdk4, Cdk4R24C, causes this kinase to be insensitive to INK4 cell cycle inhibitors and induces beta cell hyperplasia in Cdk4R24C knockin mice. We aimed to determine whether this Cdk4R24C mutation also affects proper islet function, and whether it promotes proliferation in human islets lentivirally transduced with Cdk4R24C cDNA. METHODS: Our study was conducted on wild-type and Cdk4R24C knockin mice. Pancreases were morphometrically analysed. Intraperitoneal glucose tolerance tests and intravenous insulin tolerance tests were performed on wild-type and Cdk4R24C mice. We also did in vitro islet perifusion studies and islet metabolic labelling analysis. Human islets were transduced with Cdk4R24C cDNA. RESULTS: Pancreatic islets from Cdk4R24C knockin mice exhibit a larger insulin-producing beta cell area and a higher insulin content than islets from wild-type littermates. Insulin secretion in response to glucose is faster and reaches a higher peak in Cdk4R24C mice without leading to hypoglycaemia. Conversion of pro-insulin into insulin and its intermediates is similar in Cdk4R24C and wild-type mice. Glucose utilisation and oxidation measured per islet were similar in both experimental groups. Insulin secretion was faster and enhanced in Cdk4R24C islets perifused with 16.7 mmol/l glucose, with slower decay kinetics when glucose returned to 2.8 mmol/l. Moreover, human islets expressing Cdk4R24C cDNA exhibited higher beta cell proliferation. CONCLUSIONS/INTERPRETATION: Despite their hyperplastic growth, Cdk4R24C insulin-producing islet cells behave like differentiated beta cells with regard to insulin production, insulin secretion in response to glucose, and islet glucose metabolism. Therefore Cdk4 could possibly be used to engineer a source of beta cell mass for islet transplantation.

Wide spectrum of tumors in knock-in mice carrying a Cdk4 protein insensitive to INK4 inhibitors.

We have introduced a point mutation in the first coding exon of the locus encoding the cyclin-dependent kinase 4 (Cdk4) by homologous recombination in embryonic stem cells. This mutation (replacement of Arg24 by Cys) was first found in patients with hereditary melanoma and renders Cdk4 insensitive to INK4 inhibitors. Here, we report that primary embryonic fibroblasts expressing the mutant Cdk4R24C kinase are immortal and susceptible to transformation by Ras oncogenes. Moreover, homozygous Cdk4(R24C/R24C) mutant mice develop multiple tumors with almost complete penetrance. The most common neoplasia (endocrine tumors and hemangiosarcomas) are similar to those found in pRb(+/-) and p53(-/-) mice. This Cdk4 mutation cooperates with p53 and p27(Kip1) deficiencies in decreasing tumor latency and favoring development of specific tumor types. These results provide experimental evidence for a central role of Cdk4 regulation in cancer and provide a valuable model for testing the potential anti-tumor effect of Cdk4 inhibitors in vivo.

Invasive melanoma in Cdk4-targeted mice.

Many human tumors harbor mutations that result in deregulation of Cdk4 activity. Most of these mutations involve overexpression of D-type cyclins and inactivation of INK4 inhibitors. In addition, a mutation in the Cdk4 protein has been described in patients with familial melanoma (Wolfel, T., Hauer, M., Schneider, J., Serrano, M., Wolfel, C., et al. (1995) Science 269, 1281-1284; Zuo, L., Weger, J., Yang, Q., Goldstein, A. M., Tucker, M. A., et al. (1996) Nat. Genet. 12, 97-99). This mutation, R24C, renders the Cdk4 protein insensitive to inhibition by INK4 proteins including p16(INK4a), a major candidate for the melanoma susceptibility locus. Here we show that knock-in mice expressing a Cdk4 R24C allele are highly susceptible to melanoma development after specific carcinogenic treatments. These tumors do not have mutations in the p19(ARF)/p53 pathway, suggesting a specific involvement of the p16(INK4a)/Cdk4/Rb pathway in melanoma development. Moreover, by using targeted mice deficient for other INK4 inhibitors, we show that deletion of p18(INK4c) but not of p15(INK4b) confers proliferative advantage to melanocytic tumor growth. These results provide an experimental scenario to study the role of Cdk4 regulation in melanoma and to develop novel therapeutic approaches to control melanoma progression.

trkA modulation of developing somatosensory neurons in oro-facial tissues: tooth pulp fibers are absent in trkA knockout mice.

To investigate the nerve growth factor requirement of developing oro-facial somatosensory afferents, we have studied the survival of sensory fibers subserving nociception, mechanoreception or proprioception in receptor tyrosine kinase (trkA) knockout mice using immunohistochemistry. trkA receptor null mutant mice lack nerve fibers in tooth pulp, including sympathetic fibers, and showed only sparse innervation of the periodontal ligament. Ruffini endings were formed definitively in the periodontal ligament of the trkA knockout mice, although calcitonin gene-related peptide- and substance P-immunoreactive fibers were reduced in number or had disappeared completely. trkA gene deletion had also no obvious effect on the formation of Meissner corpuscles in the palate. In the vibrissal follicle, however, some mechanoreceptive afferents were sensitive for trkA gene deletion, confirming a previous report [Fundin et al. (1997) Dev. Biol. 190, 94-116]. Moreover, calretinin-positive fibers innervating longitudinal lanceolate endings were completely lost in trkA knockout mice, as were the calretinin-containing parent cells in the trigeminal ganglion.These results indicate that trkA is indispensable for developing nociceptive neurons innervating oral tissues, but not for developing mechanoreceptive neurons innervating oral tissues (Ruffini endings and Meissner corpuscles), and that calretinin-containing, trkA dependent neurons in the trigeminal ganglion normally participate in mechanoreception through longitudinal lanceolate endings of the vibrissal follicle.

Compensation between Vav-1 and Vav-2 in B cell development and antigen receptor signaling.

Vav-1 and Vav-2 are closely related Dbl-homology GTP exchange factors (GEFs) for Rho GTPases. Mutation of Vav-1 disrupts T cell development and T cell antigen receptor-induced activation, but has comparatively little effect on B cells. We found that combined deletion of both Vav-1 and Vav-2 in mice resulted in a marked reduction in mature B lymphocyte numbers. Vav-1(-/-)Vav-2(-/-) B cells were unresponsive to B cell antigen receptor (BCR)-driven proliferation in vitro and to thymus-independent antigen in vivo. BCR-stimulated intracellular calcium mobilization was greatly impaired in Vav-1(-/-)Vav-2(-/-) B cells. These findings establish a role for Vav-2 in BCR calcium signaling and reveal that the Vav family of GEFs is critical to B cell development and function.

Control of spermatogenesis in mice by the cyclin D-dependent kinase inhibitors p18(Ink4c) and p19(Ink4d).

Male mice lacking both the Ink4c and Ink4d genes, which encode two inhibitors of D-type cyclin-dependent kinases (Cdks), are infertile, whereas female fecundity is unaffected. Both p18(Ink4c) and p19(Ink4d) are expressed in the seminiferous tubules of postnatal wild-type mice, being largely confined to postmitotic spermatocytes undergoing meiosis. Their combined loss is associated with the delayed exit of spermatogonia from the mitotic cell cycle, leading to the retarded appearance of meiotic cells that do not properly differentiate and instead undergo apoptosis at an increased frequency. As a result, mice lacking both Ink4c and Ink4d produce few mature sperm, and the residual spermatozoa have reduced motility and decreased viability. Whether or not Ink4d is present, animals lacking Ink4c develop hyperplasia of interstitial testicular Leydig cells, which produce reduced levels of testosterone. The anterior pituitary of fertile mice lacking Ink4c or infertile mice doubly deficient for Ink4c and Ink4d produces normal levels of luteinizing hormone (LH). Therefore, the failure of Leydig cells to produce testosterone is not secondary to defects in LH production, and reduced testosterone levels do not account for infertility in the doubly deficient strain. By contrast, Ink4d-null or double-null mice produce elevated levels of follicle-stimulating hormone (FSH). Because Ink4d-null mice are fertile, increased FSH production by the anterior pituitary is also unlikely to contribute to the sterility observed in Ink4c/Ink4d double-null males. Our data indicate that p18(Ink4c) and p19(Ink4d) are essential for male fertility. These two Cdk inhibitors collaborate in regulating spermatogenesis, helping to ensure mitotic exit and the normal meiotic maturation of spermatocytes.

Effects of gamma-linolenic acid and oleic acid on paclitaxel cytotoxicity in human breast cancer cells.

It has been suggested that dietary interventions may improve the effectiveness of cancer chemotherapy. We have examined the combined in vitro cytotoxicity of paclitaxel and the fatty acids gamma-linolenic acid (GLA, 18:3n-6) and oleic acid (OA, 18:1n-9) in human breast carcinoma MDA-MB-231 cells. The effect of fatty acids on paclitaxel chemosensitivity was determined by comparing IC(50) and IC(70) (50 and 70% inhibitory concentrations, respectively) obtained when the cells were exposed to IC(50) and IC(70) levels of paclitaxel alone and fatty acids were supplemented either before or during the exposure to paclitaxel. The 3-4,5-dimethylthiazol-2-yl-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to determine cell growth inhibition. GLA by itself showed antiproliferative effects, and a possible GLA-paclitaxel interaction at the cellular level was assessed by the isobologram and the combination-index (CI) methods. Isobole analysis at the isoeffect levels of 50 and 70% revealed that drug interaction was predominantly synergistic when GLA and paclitaxel were added concurrently for 24 h to the cell cultures. Interaction assessment using the median-effect principle and the combination-index (CI) method showed that exposure of MDA-MB-231 cells to an equimolar combination of concurrent GLA plus paclitaxel for 24 h resulted in a moderate synergism at all effect levels, consistent with the results of the isobologram analysis. When exposure to GLA (24 h) was followed sequentially by paclitaxel (24 h) only an additive effect was observed. The GLA-mediated increase in paclitaxel chemosensitivity was only partially abolished by Vitamin E, a lipid peroxidation inhibitor, suggesting a limited influence of the oxidative status of GLA in achieving potentiation of paclitaxel toxicity. When OA (a non-peroxidisable fatty acid) was combined with paclitaxel, an enhancement of chemosensitivity was found when OA was used concurrently with paclitaxel, although less markedly than with GLA. Pretreatment of MDA-MB-231 cells with OA for 24 h prior to a 24 h paclitaxel exposure produced greater enhancement of paclitaxel sensitivity at high OA concentrations than the concurrent exposure to OA and paclitaxel. The OA-induced sensitisation to paclitaxel was not due to the cytoxicity of the fatty acid itself. When these observations were extended to three additional breast carcinoma cell lines (SK-Br3, T47D and MCF-7), simultaneous exposure to GLA and paclitaxel also resulted in synergism. GLA preincubation followed by paclitaxel resulted in additivity for all cell lines. Simultaneous exposure to paclitaxel and OA enhanced paclitaxel cytotoxicity in T47D and MCF-7 cells, but not in SK-Br3 cells, whereas preincubation with OA failed to increase paclitaxel effectiveness in all three cell lines. For comparison, the effects of other fatty acids on paclitaxel chemosensitivity were examined: GLA was the most potent at enhancing paclitaxel cytotoxicity, followed by alpha-linolenic acid (ALA; 18:3n.3), eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3), whereas linoleic acid (LA; 18:2n-6) did not increase paclitaxel toxicity. These findings provide experimental support for the use of fatty acids as modulators of tumour cell chemosensitivity in paclitaxel-based therapy.

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.

Genetic analysis of mammalian cyclin-dependent kinases and their inhibitors.

Entry into the cell cycle, in particular the G1/S transition, is a tightly regulated process that involves a combination of mitogenic signaling pathways and cell cycle checkpoints. Some of the key regulators of this process are frequently altered in human cancer. Although the proteins that control the G1/S transition have been extensively studied at the biochemical level, little is known regarding their physiological role in vivo. During the last few years, a series of mouse strains carrying gene targeted mutations in key regulators of the G1/S transition have been generated. They include the Rb family of proteins and some of their downstream and upstream regulators. The latter include the regulatory (cyclin) and catalytic (Cdk) subunits of some of the kinases responsible for Rb inactivation as well as all the members of two families of cell cycle inhibitors, the INK4 and the Cip/Kip proteins. In this review, we summarize the most relevant information derived from the characterization of these strains of mice and attempt to integrate it within a functional framework of cell cycle regulation in vivo.

Limited overlapping roles of P15(INK4b) and P18(INK4c) cell cycle inhibitors in proliferation and tumorigenesis.

Entry of quiescent cells into the cell cycle is driven by the cyclin D-dependent kinases Cdk4 and Cdk6. These kinases are negatively regulated by the INK4 cell cycle inhibitors. We report the generation of mice defective in P15(INK4b) and P18(INK4c). Ablation of these genes, either alone or in combination, does not abrogate cell contact inhibition or senescence of mouse embryo fibroblasts in culture. However, loss of P15(INK4b), but not of P18(INK4c), confers proliferative advantage to these cells and makes them more sensitive to transformation by H-ras oncogenes. In vivo, ablation of P15(INK4b) and P18(INK4c) genes results in lymphoproliferative disorders and tumor formation. Mice lacking P18(INK4c) have deregulated epithelial cell growth leading to the formation of cysts, mostly in the cortical region of the kidneys and the mammary epithelium. Loss of both P15(INK4b) and P18(INK4c) does not result in significantly distinct phenotypic manifestations except for the appearance of cysts in additional tissues. These results indicate that P15(INK4b) and P18(IKN4c) are tumor suppressor proteins that act in different cellular lineages and/or pathways with limited compensatory roles.

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.