Identification of cancer initiating cells in K-Ras driven lung adenocarcinoma.

Ubiquitous expression of a resident K-Ras(G12V) oncogene in adult mice revealed that most tissues are resistant to K-Ras oncogenic signals. Indeed, K-Ras(G12V) expression only induced overt tumors in lungs. To identify these transformation-permissive cells, we induced K-Ras(G12V) expression in a very limited number of adult lung cells (0.2%) and monitored their fate by X-Gal staining, a surrogate marker coexpressed with the K-Ras(G12V) oncoprotein. Four weeks later, 30% of these cells had proliferated to form small clusters. However, only SPC(+) alveolar type II (ATII) cells were able to form hyperplastic lesions, some of which progressed to adenomas and adenocarcinomas. In contrast, induction of K-Ras(G12V) expression in lung cells by intratracheal infection with adenoviral-Cre particles generated hyperplasias in all regions except the proximal airways. Bronchiolar and bronchioalveolar duct junction hyperplasias were primarily made of CC10(+) Clara cells. Some of them progressed to form benign adenomas. However, only alveolar hyperplasias, exclusively made up of SPC(+) ATII cells, progressed to yield malignant adenocarcinomas. Adenoviral infection induced inflammatory infiltrates primarily made of T and B cells. This inflammatory response was essential for the development of K-Ras(G12V)-driven bronchiolar hyperplasias and adenomas, but not for the generation of SPC(+) ATII lesions. Finally, activation of K-Ras(G12V) during embryonic development under the control of a Sca1 promoter yielded CC10(+), but not SPC(+), hyperplasias, and adenomas. These results, taken together, illustrate that different types of lung cells can generate benign lesions in response to K-Ras oncogenic signals. However, in adult mice, only SPC(+) ATII cells were able to yield malignant adenocarcinomas.

Inactivation of the p53 pathway in retinoblastoma.

Most human tumours have genetic mutations in their Rb and p53 pathways, but retinoblastoma is thought to be an exception. Studies suggest that retinoblastomas, which initiate with mutations in the gene retinoblastoma 1 (RB1), bypass the p53 pathway because they arise from intrinsically death-resistant cells during retinal development. In contrast to this prevailing theory, here we show that the tumour surveillance pathway mediated by Arf, MDM2, MDMX and p53 is activated after loss of RB1 during retinogenesis. RB1-deficient retinoblasts undergo p53-mediated apoptosis and exit the cell cycle. Subsequently, amplification of the MDMX gene and increased expression of MDMX protein are strongly selected for during tumour progression as a mechanism to suppress the p53 response in RB1-deficient retinal cells. Our data provide evidence that the p53 pathway is inactivated in retinoblastoma and that this cancer does not originate from intrinsically death-resistant cells as previously thought. In addition, they support the idea that MDMX is a specific chemotherapeutic target for treating retinoblastoma.

c-RAF Ablation Induces Regression of Advanced Kras/Trp53 Mutant Lung Adenocarcinomas by a Mechanism Independent of MAPK Signaling.

A quarter of all solid tumors harbor KRAS oncogenes. Yet, no selective drugs have been approved to treat these malignancies. Genetic interrogation of the MAPK pathway revealed that systemic ablation of MEK or ERK kinases in adult mice prevent tumor development but are unacceptably toxic. Here, we demonstrate that ablation of c-RAF expression in advanced tumors driven by KrasG12V/Trp53 mutations leads to significant tumor regression with no detectable appearance of resistance mechanisms. Tumor regression results from massive apoptosis. Importantly, systemic abrogation of c-RAF expression does not inhibit canonical MAPK signaling, hence, resulting in limited toxicities. These results are of significant relevance for the design of therapeutic strategies to treat K-RAS mutant cancers.

Amplification of Mdmx (or Mdm4) directly contributes to tumor formation by inhibiting p53 tumor suppressor activity.

Human tumors are believed to harbor a disabled p53 tumor suppressor pathway, either through direct mutation of the p53 gene or through aberrant expression of proteins acting in the p53 pathway, such as p14(ARF) or Mdm2. A role for Mdmx (or Mdm4) as a key negative regulator of p53 function in vivo has been established. However, a direct contribution of Mdmx to tumor formation remains to be demonstrated. Here we show that retrovirus-mediated Mdmx overexpression allows primary mouse embryonic fibroblast immortalization and leads to neoplastic transformation in combination with HRas(V12). Furthermore, the human Mdmx ortholog, Hdmx, was found to be overexpressed in a significant percentage of various human tumors and amplified in 5% of primary breast tumors, all of which retained wild-type p53. Hdmx was also amplified and highly expressed in MCF-7, a breast cancer cell line harboring wild-type p53, and interfering RNA-mediated reduction of Hdmx markedly inhibited the growth potential of these cells in a p53-dependent manner. Together, these results make Hdmx a new putative drug target for cancer therapy.

[The mouse as preclinical models of lung cancer]. / Modèles précliniques chez le rongeur de carcinomes pulmonaires.

The high incidence and poor prognosis of lung cancer represent a major health problem. Currently, about 20% of lung cancer patients can benefit from targeted therapy after identification of EGFR, ALK or HER2 somatic mutations or rearrangements. Other mutations, such as KRas oncogenic mutation, are still orphans of validated targeted therapy. In this review, we describe the different mouse models of lung carcinoma. We then illustrate the interests of such models for the identification and validation of new therapeutic targets, for the study of secondary resistance and for their use as preclinical models and for new therapeutic strategy tests.

c-Raf, but not B-Raf, is essential for development of K-Ras oncogene-driven non-small cell lung carcinoma.

We have investigated the role of individual members of the Raf/Mek/Erk cascade in the onset of K-Ras oncogene-driven non-small cell lung carcinoma (NSCLC). Ablation of Erk1 or Erk2 in K-Ras oncogene-expressing lung cells had no significant effect due to compensatory activities. Yet, elimination of both Erk kinases completely blocked tumor development. Similar results were obtained with Mek kinases. Ablation of B-Raf had no significant effect on tumor development. However, c-Raf expression was absolutely essential for the onset of NSCLC. Interestingly, concomitant elimination of c-Raf and B-Raf in adult mice had no deleterious consequences for normal homeostasis. These results indicate that c-Raf plays a unique role in mediating K-Ras signaling and makes it a suitable target for therapeutic intervention.

Distinct roles of Mdm2 and Mdm4 in red cell production.

Mdm2 and Mdm4 are critical negative regulators of the p53 tumor suppressor. Mdm4-null mutants are severely anemic and exhibit impaired proliferation of the fetal liver erythroid lineage cells. This phenotype may indicate a cell-intrinsic function of Mdm4 in erythropoiesis. In contrast, red blood cell count was nearly normal in mice engineered to express low levels of Mdm2, suggesting that Mdm2 might be dispensable for red cell production. Here, we further explore the tissue-specific functions of Mdm2 and Mdm4 in the erythroid lineage by intercrossing conditional Mdm4 and Mdm2 alleles to an erythroid-specific Cre (Er-GFP-Cre) knock-in allele. Our data show that Mdm2 is required for rescuing erythroid progenitors from p53-mediated apoptosis during primitive erythropoiesis. In contrast, Mdm4 is only required for the high erythropoietic rate during embryonic definitive erythropoiesis. Thus, in this particular cellular context, Mdm4 only contributes to p53 regulation at a specific phase of the differentiation program.

A critical role for p53 in the control of NF-kappaB-dependent gene expression in TLR4-stimulated dendritic cells exposed to Genistein.

Considerable research has focused on the anti-inflammatory and antiproliferative activities exhibited by the soy isoflavone genistein. We previously demonstrated that genistein suppresses TNF-alpha-induced NF-kappaB-dependent IL-6 gene expression in cancer cells by interfering with the mitogen- and stress-activated protein kinase 1 activation pathway. However, effects of isoflavones on immune cells, such as dendritic cells, remain largely unknown. Here we show that genistein markedly reduces IL-6 cytokine production and transcription in LPS-stimulated human monocyte-derived dendritic cells. More particularly, we observe that genistein inhibits IL-6 gene expression by modulating the transcription factor NF-kappaB. Examination of NF-kappaB-related events downstream of TLR4 demonstrates that genistein affects NF-kappaB subcellular localization and DNA binding, although we observe only a minor inhibitory impact of genistein on the classical LPS-induced signaling steps. Interestingly, we find that genistein significantly increases p53 protein levels. We also show that overexpression of p53 in TLR4/MD2 HEK293T cells blocks LPS-induced NF-kappaB-dependent gene transcription, indicating the occurrence of functional cross-talk between p53 and NF-kappaB. Moreover, analysis of IL-6 mRNA levels in bone marrow-derived p53 null vs wild-type dendritic cells confirms a role for p53 in the reduction of NF-kappaB-dependent gene expression, mediated by genistein.

Mdm4 and Mdm2 cooperate to inhibit p53 activity in proliferating and quiescent cells in vivo.

The Mdm2 and Mdm4 oncoproteins are key negative regulators of the p53 tumor suppressor. However, their physiological contributions to the regulation of p53 stability and activity remain highly controversial. Here, we combined a p53 knock-in allele, in which p53 is silenced by a transcriptional stop element flanked by loxP sites, with the mdm2- and mdm4-null alleles. This approach allows Cre-mediated conditional p53 expression in tissues in vivo and cells in vitro lacking Mdm2, Mdm4, or both. Using this strategy, we show that Mdm2 and Mdm4 are essential in a nonredundant manner for preventing p53 activity in the same cell type, irrespective of the proliferation/differentiation status of the cells. Although Mdm2 prevents accumulation of the p53 protein, Mdm4 contributes to the overall inhibition of p53 activity independent of Mdm2. We propose a model in which Mdm2 is critical for the regulation of p53 levels and Mdm4 is critical for the fine-tuning of p53 transcriptional activity, both proteins acting synergistically to keep p53 in check.