Carmen Guerra

Tumour biology: Senescence in premalignant tumours

Oncogene-induced senescence is a cellular response that may be crucial for protection against cancer development, but its investigation has so far been restricted to cultured cells that have been manipulated to overexpress an oncogene. Here we analyse tumours initiated by an endogenous oncogene, ras, and show that senescent cells exist in premalignant tumours but not in malignant ones. Senescence is therefore a defining feature of premalignant tumours that could prove valuable in the diagnosis and prognosis of cancer.

Emergence of brown adipocytes in white fat in mice is under genetic control. Effects on body weight and adiposity.

The mRNA levels for the mitochondrial uncoupling protein (UCP1) in fat tissues of A/J and C57BL/6J inbred strains of mice varied in a regional-specific manner after stimulation of adrenergic signaling by cold exposure or treatment with a beta3-adrenergic agonist. While the differences between strains were minimal in interscapular brown fat, large differences occurred in white fat tissues, particularly in retroperitoneal fat. Among the AXB recombinant inbred strains, the Ucp1 mRNA levels varied up to 130-fold. This large induction at the mRNA level was accompanied by a corresponding increase in brown adipocytes as revealed by immunohistology with anti-UCP1 antibodies. A high capacity to induce brown fat in areas of traditional white fat had no impact on the ability to gain weight in response to high fat and sucrose diets, but did correlate with the loss of weight in response to treatment with a beta3-adrenergic agonist (CL 316,243). This genetic variation in mice provides an experimental approach to identify genes controlling the induction of brown adipocytes in white fat tissues.

Tumor induction by an endogenous K-ras oncogene is highly dependent on cellular context

We have targeted a K-ras allele in mouse embryonic stem (ES) cells to express a K-Ras(V12) oncoprotein along with a marker protein (beta-geo) from a single bicistronic transcript. Expression of this oncogenic allele requires removal of a knocked in STOP transcriptional cassette by Cre recombinase. Primary mouse embryonic fibroblasts expressing this K-ras(V12) allele do not undergo proliferative senescence and proliferate as immortal cells. In mice, expression of K-ras(V12) throughout the body fails to induce unscheduled proliferation or other growth abnormalities for up to eight months. Only a percentage of K-ras(V12)-expressing lung bronchiolo-alveolar cells undergo malignant transformation leading to the formation of multiple adenomas and adenocarcinomas. These results indicate that neoplastic growth induced by an endogenous K-ras oncogene depends upon cellular context.

Pancreatitis-Induced Inflammation Contributes to Pancreatic Cancer by Inhibiting Oncogene-Induced Senescence

Pancreatic acinar cells of adult mice (≥P60) are resistant to transformation by some of the most robust oncogenic insults including expression of K-Ras oncogenes and loss of p16Ink4a/p19Arf or Trp53 tumor suppressors. Yet, these acinar cells yield pancreatic intraepithelial neoplasias (mPanIN) and ductal adenocarcinomas (mPDAC) if exposed to limited bouts of non-acute pancreatitis, providing they harbor K-Ras oncogenes. Pancreatitis contributes to tumor progression by abrogating the senescence barrier characteristic of low-grade mPanINs. Attenuation of pancreatitis-induced inflammation also accelerates tissue repair and thwarts mPanIN expansion. Patients with chronic pancreatitis display senescent PanINs, providing they have received antiinflammatory drugs. These results support the concept that antiinflammatory treatment of people diagnosed with pancreatitis may reduce their risk of developing PDAC.

p38α MAP kinase is essential in lung stem and progenitor cell proliferation and differentiation

Stem cell function is central for the maintenance of normal tissue homeostasis. Here we show that deletion of p38α mitogen-activated protein (MAP) kinase in adult mice results in increased proliferation and defective differentiation of lung stem and progenitor cells both in vivo and in vitro. We found that p38α positively regulates factors such as CCAAT/enhancer-binding protein that are required for lung cell differentiation. In addition, p38α controls self-renewal of the lung stem and progenitor cell population by inhibiting proliferation-inducing signals, most notably epidermal growth factor receptor. As a consequence, the inactivation of p38α leads to an immature and hyperproliferative lung epithelium that is highly sensitized to K-RasG12V-induced tumorigenesis. Our results indicate that by coordinating proliferation and differentiation signals in lung stem and progenitor cells, p38α has a key role in the regulation of lung cell renewal and tumorigenesis.

A Synthetic Lethal Interaction between K-Ras Oncogenes and Cdk4 Unveils a Therapeutic Strategy for Non-small Cell Lung Carcinoma

We have unveiled a synthetic lethal interaction between K-Ras oncogenes and Cdk4 in a mouse tumor model that closely recapitulates human non-small cell lung carcinoma (NSCLC). Ablation of Cdk4, but not Cdk2 or Cdk6, induces an immediate senescence response only in lung cells that express an endogenous K-Ras oncogene. No such response occurs in lungs expressing a single Cdk4 allele or in other K-Ras-expressing tissues. More importantly, targeting Cdk4 alleles in advanced tumors detectable by computed tomography scanning also induces senescence and prevents tumor progression. These observations suggest that robust and selective pharmacological inhibition of Cdk4 may provide therapeutic benefit for NSCLC patients carrying K-RAS oncogenes.

Exploiting oncogene-induced replicative stress for the selective killing of Myc-driven tumors

Oncogene-induced replicative stress activates an Atr- and Chk1-dependent response, which has been proposed to be widespread in tumors. We explored whether the presence of replicative stress could be exploited for the selective elimination of cancer cells. To this end, we evaluated the impact of targeting the replicative stress-response on cancer development. In mice (Mus musculus), the reduced levels of Atr found on a mouse model of the Atr-Seckel syndrome completely prevented the development of Myc-induced lymphomas or pancreatic tumors, both of which showed abundant levels of replicative stress. Moreover, Chk1 inhibitors were highly effective in killing Myc-driven lymphomas. By contrast, pancreatic adenocarcinomas initiated by K-RasG12V showed no detectable evidence of replicative stress and were nonresponsive to this therapy. Besides its impact on cancer, Myc overexpression aggravated the phenotypes of Atr-Seckel mice, revealing that oncogenes can modulate the severity of replicative stress-associated diseases.

Brown adipose tissue–specific insulin receptor knockout shows diabetic phenotype without insulin resistance

Although insulin regulates metabolism in both brown and white adipocytes, the role of these tissues in energy storage and utilization is quite different. Recombination technology using the Cre-loxP approach allows inactivation of the insulin receptor in a tissue-specific manner. Mice lacking insulin receptors in brown adipocytes show an age-dependent loss of interscapular brown fat but increased expression of uncoupling protein-1 and -2. In parallel, these mice develop an insulin-secretion defect resulting in a progressive glucose intolerance, without insulin resistance. This model provides direct evidence for not only a role for the insulin receptors in brown fat adipogenesis, the data also suggest a novel role of brown adipose tissue in the regulation of insulin secretion and glucose homeostasis.

Loss of Apc allows phenotypic manifestation of the transforming properties of an endogenous K-ras oncogene in vivo

Oncogenic mutations in the K-ras gene occur in ≈50% of human colorectal cancers. However, the precise role that K-ras oncogenes play in tumor formation is still unclear. To address this issue, we have conditionally expressed an oncogenic K-rasV12 allele in the small intestine of adult mice either alone or in the context of Apc deficiency. We found that expression of K-rasV12 does not affect normal intestinal homeostasis or the immediate phenotypes associated with Apc deficiency. Mechanistically we failed to find activation of the Raf/MEK/ERK pathway, which may be a consequence of the up-regulation of a number of negative feedback loops. However, K-rasV12 expression accelerates intestinal tumorigenesis and confers invasive properties after Apc loss over the long term. In renal epithelium, expression of the oncogenic K-rasV12 allele in the absence of Apc induces the rapid development of renal carcinoma. These tumors, unlike those of intestinal origin, display activation of the Raf/MEK/ERK and Akt signaling pathways. Taken together, these data indicate that normal intestinal and kidney epithelium are resistant to malignant transformation by an endogenous K-ras oncogene. However, activation of K-rasV12 after Apc loss results in increased tumorigenesis with distinct kinetics. Whereas the effect of K-ras oncogenes in the intestine can been observed only after long latencies, they result in rapid carcinogenesis in the kidney epithelium. These data imply a window of opportunity for anti-K-ras therapies after tumor initiation in preventing tumor growth and invasion.

Genetic analysis of Ras signalling pathways in cell proliferation, migration and survival

We have used mouse embryonic fibroblasts (MEFs) devoid of Ras proteins to illustrate that they are essential for proliferation and migration, but not for survival, at least in these cells. These properties are unique to the Ras subfamily of proteins because ectopic expression of other Ras‐like small GTPases, even when constitutively active, could not compensate for the absence of Ras proteins. Only constitutive activation of components of the Raf/Mek/Erk pathway was sufficient to sustain normal proliferation and migration of MEFs devoid of Ras proteins. Activation of the phosphatidylinositol 3‐kinase (PI3K)/PTEN/Akt and Ral guanine exchange factor (RalGEF)/Ral pathways, either alone or in combination, failed to induce proliferation or migration of Rasless cells, although they cooperated with Raf/Mek/Erk signalling to reproduce the full response mediated by Ras signalling. In contrast to current hypotheses, Ras signalling did not induce proliferation by inducing expression of D‐type Cyclins. Rasless MEFs had normal levels of Cyclin D1/Cdk4 and Cyclin E/Cdk2. However, these complexes were inactive. Inactivation of the pocket proteins or knock down of pRb relieved MEFs from their dependence on Ras signalling to proliferate.