A mouse model for Li-Fraumeni-Like Syndrome with cardiac angiosarcomas associated to POT1 mutations.

The shelterin protein POT1 has been found mutated in many different familial and sporadic cancers, however, no mouse models to understand the pathobiology of these mutations have been developed so far. To address the molecular mechanisms underlying the tumorigenic effects of POT1 mutant proteins in humans, we have generated a mouse model for the human POT1R117C mutation found in Li-Fraumeni-Like families with cases of cardiac angiosarcoma by introducing this mutation in the Pot1a endogenous locus, knock-in for Pot1aR117C. We find here that both mouse embryonic fibroblasts (MEFs) and tissues from Pot1a+/ki mice show longer telomeres than wild-type controls. Longer telomeres in Pot1a+/ki MEFs are dependent on telomerase activity as they are not found in double mutant Pot1a+/ki Tert-/- telomerase-deficient MEFs. By using complementation assays we further show that POT1a pR117C exerts dominant-negative effects at telomeres. As in human Li-Fraumeni patients, heterozygous Pot1a+/ki mice spontaneously develop a high incidence of angiosarcomas, including cardiac angiosarcomas, and this is associated to the presence of abnormally long telomeres in endothelial cells as well as in the tumors. The Pot1a+/R117C mouse model constitutes a useful tool to understand human cancers initiated by POT1 mutations.

Stromal oncostatin M cytokine promotes breast cancer progression by reprogramming the tumor microenvironment.

The tumor microenvironment (TME) is reprogrammed by cancer cells and participates in all stages of tumor progression. The contribution of stromal cells to the reprogramming of the TME is not well understood. Here, we provide evidence of the role of the cytokine oncostatin M (OSM) as central node for multicellular interactions between immune and nonimmune stromal cells and the epithelial cancer cell compartment. OSM receptor (OSMR) deletion in a multistage breast cancer model halted tumor progression. We ascribed causality to the stromal function of the OSM axis by demonstrating reduced tumor burden of syngeneic tumors implanted in mice lacking OSMR. Single-cell and bioinformatic analysis of murine and human breast tumors revealed that OSM expression was restricted to myeloid cells, whereas OSMR was detected predominantly in fibroblasts and, to a lower extent, cancer cells. Myeloid-derived OSM reprogrammed fibroblasts to a more contractile and tumorigenic phenotype and elicited the secretion of VEGF and proinflammatory chemokines CXCL1 and CXCL16, leading to increased myeloid cell recruitment. Collectively, our data support the notion that the stromal OSM/OSMR axis reprograms the immune and nonimmune microenvironment and plays a key role in breast cancer progression.

Genomic and Functional Regulation of TRIB1 Contributes to Prostate Cancer Pathogenesis.

Prostate cancer is the most frequent malignancy in European men and the second worldwide. One of the major oncogenic events in this disease includes amplification of the transcription factor cMYC. Amplification of this oncogene in chromosome 8q24 occurs concomitantly with the copy number increase in a subset of neighboring genes and regulatory elements, but their contribution to disease pathogenesis is poorly understood. Here we show that TRIB1 is among the most robustly upregulated coding genes within the 8q24 amplicon in prostate cancer. Moreover, we demonstrate that TRIB1 amplification and overexpression are frequent in this tumor type. Importantly, we find that, parallel to its amplification, TRIB1 transcription is controlled by cMYC. Mouse modeling and functional analysis revealed that aberrant TRIB1 expression is causal to prostate cancer pathogenesis. In sum, we provide unprecedented evidence for the regulation and function of TRIB1 in prostate cancer.

Genetic manipulation of LKB1 elicits lethal metastatic prostate cancer.

Gene dosage is a key defining factor to understand cancer pathogenesis and progression, which requires the development of experimental models that aid better deconstruction of the disease. Here, we model an aggressive form of prostate cancer and show the unconventional association of LKB1 dosage to prostate tumorigenesis. Whereas loss of Lkb1 alone in the murine prostate epithelium was inconsequential for tumorigenesis, its combination with an oncogenic insult, illustrated by Pten heterozygosity, elicited lethal metastatic prostate cancer. Despite the low frequency of LKB1 deletion in patients, this event was significantly enriched in lung metastasis. Modeling the role of LKB1 in cellular systems revealed that the residual activity retained in a reported kinase-dead form, LKB1K78I, was sufficient to hamper tumor aggressiveness and metastatic dissemination. Our data suggest that prostate cells can function normally with low activity of LKB1, whereas its complete absence influences prostate cancer pathogenesis and dissemination.

SOX9 promotes tumor progression through the axis BMI1-p21CIP.

The developmental regulator SOX9 is linked to cancer progression mainly as a result of its role in the regulation of cancer stem cells (CSCs). However, its activity in the differentiated cells that constitute the heterogeneous tumor bulk has not been extensively studied. In this work, we addressed this aspect in gastric cancer, glioblastoma and pancreatic adenocarcinoma. SOX9 silencing studies revealed that SOX9 is required for cancer cell survival, proliferation and evasion of senescence in vitro and tumor growth in vivo. Gain of-SOX9 function showed that high levels of SOX9 promote tumor cell proliferation in vitro and in vivo. Mechanistically, the modulation of SOX9 changed the expression of the transcriptional repressor BMI1 in the same direction in the three types of cancer, and the expression of the tumor suppressor p21CIP in the opposite direction. In agreement with this, SOX9 expression positively correlated with BMI1 levels and inversely with p21CIP in clinical samples of the different cancers. Moreover, BMI1 re-establishment in SOX9-silenced tumor cells restored cell viability and proliferation as well as decreased p21CIP in vitro and tumor growth in vivo. These results indicate that BMI1 is a critical effector of the pro-tumoral activity of SOX9 in tumor bulk cells through the repression of p21CIP. Our results highlight the relevance of the SOX9-BMI1-p21CIP axis in tumor progression, shedding novel opportunities for therapeutic development.

Safety of Whole-Body Abrogation of the TRF1 Shelterin Protein in Wild-Type and Cancer-Prone Mouse Models.

Telomeres are considered potential anti-cancer targets. Most studies have focused on telomerase inhibition, but this strategy has largely failed in clinical trials. Direct disruption of the shelterin complex through TRF1 inhibition can block tumorigenesis in cancer mouse models by a mechanism that involves DNA damage induction and reduction of proliferation and stemness. Any anti-cancer target, however, must fulfill the requisite of not showing deleterious effects in healthy tissues. Here, we show that Trf1 genetic deletion in wild-type and cancer-prone p53- and Ink4Arf-deficient mice does not affect organismal viability and only induces mild phenotypes like decreased body weight and hair graying or hair loss, the skin being the most affected tissue. Importantly, we found that Trf1 is essential for tumorigenesis in p53- and Ink4Arf-deficient mice, as we did not find a single tumor originating from Trf1-deleted cells. These findings indicate a therapeutic window for targeting Trf1 in cancer treatment.

Protective Role of Hepatocyte Cyclooxygenase-2 Expression Against Liver Ischemia-Reperfusion Injury in Mice.

Liver ischemia and reperfusion injury (IRI) remains a serious clinical problem affecting liver transplantation outcomes. IRI causes up to 10% of early organ failure and predisposes to chronic rejection. Cyclooxygenase-2 (COX-2) is involved in different liver diseases, but the significance of COX-2 in IRI is a matter of controversy. This study was designed to elucidate the role of COX-2 induction in hepatocytes against liver IRI. In the present work, hepatocyte-specific COX-2 transgenic mice (hCOX-2-Tg) and their wild-type (Wt) littermates were subjected to IRI. hCOX-2-Tg mice exhibited lower grades of necrosis and inflammation than Wt mice, in part by reduced hepatic recruitment and infiltration of neutrophils, with a concomitant decrease in serum levels of proinflammatory cytokines. Moreover, hCOX-2-Tg mice showed a significant attenuation of the IRI-induced increase in oxidative stress and hepatic apoptosis, an increase in autophagic flux, and a decrease in endoplasmic reticulum stress compared to Wt mice. Interestingly, ischemic preconditioning of Wt mice resembles the beneficial effects observed in hCOX-2-Tg mice against IRI due to a preconditioning-derived increase in endogenous COX-2, which is mainly localized in hepatocytes. Furthermore, measurement of prostaglandin E2 (PGE2 ) levels in plasma from patients who underwent liver transplantation revealed a significantly positive correlation of PGE2 levels and graft function and an inverse correlation with the time of ischemia. Conclusion: These data support the view of a protective effect of hepatic COX-2 induction and the consequent rise of derived prostaglandins against IRI.

Low-dose statin treatment increases prostate cancer aggressiveness.

Prostate cancer is diagnosed late in life, when co-morbidities are frequent. Among them, hypertension, hypercholesterolemia, diabetes or metabolic syndrome exhibit an elevated incidence. In turn, prostate cancer patients frequently undergo chronic pharmacological treatments that could alter disease initiation, progression and therapy response. Here we show that treatment with anti-cholesterolemic drugs, statins, at doses achieved in patients, enhance the pro-tumorigenic activity of obesogenic diets. In addition, the use of a mouse model of prostate cancer and human prostate cancer xenografts revealed that in vivo simvastatin administration alone increases prostate cancer aggressiveness. In vitro cell line systems supported the notion that this phenomenon occurs, at least in part, through the direct action on cancer cells of low doses of statins, in range of what is observed in human plasma. In sum, our results reveal a prostate cancer experimental system where statins exhibit an undesirable effect, and warrant further research to address the relevance and implications of this observation in human prostate cancer.

Constitutive Cyclin O deficiency results in penetrant hydrocephalus, impaired growth and infertility.

Cyclin O (encoded by CCNO) is a member of the cyclin family with regulatory functions in ciliogenesis and apoptosis. Homozygous CCNO mutations have been identified in human patients with Reduced Generation of Multiple Motile Cilia (RGMC) and conditional inactivation of Ccno in the mouse recapitulates some of the pathologies associated with the human disease. These include defects in the development of motile cilia and hydrocephalus. To further investigate the functions of Ccno in vivo, we have generated a new mouse model characterized by the constitutive loss of Ccno in all tissues and followed a cohort during ageing. Ccno-/- mice were growth impaired and developed hydrocephalus with high penetrance. In addition, some Ccno+/- mice also developed hydrocephalus and affected Ccno-/- and Ccno+/- mice exhibited additional CNS defects including cortical thinning and hippocampal abnormalities. In addition to the CNS defects, both male and female Ccno-/- mice were infertile and female mice exhibited few motile cilia in the oviduct. Our results further establish CCNO as an important gene for normal development and suggest that heterozygous CCNO mutations could underlie hydrocephalus or diminished fertility in some human patients.

Targeted depletion of PIK3R2 induces regression of lung squamous cell carcinoma.

Oncogenic mutations in the PI3K/AKT pathway are present in nearly half of human tumors. Nonetheless, inhibitory compounds of the pathway often induce pathway rebound and tumor resistance. We find that lung squamous cell carcinoma (SQCC), which accounts for ~20% of lung cancer, exhibits increased expression of the PI3K subunit PIK3R2, which is at low expression levels in normal tissues. We tested a new approach to interfere with PI3K/AKT pathway activation in lung SQCC. We generated tumor xenografts of SQCC cell lines and examined the consequences of targeting PIK3R2 expression. In tumors with high PIK3R2 expression, and independently of PIK3CA, KRAS, or PTEN mutations, PIK3R2 depletion induced lung SQCC xenograft regression without triggering PI3K/AKT pathway rebound. These results validate the use PIK3R2 interfering tools for the treatment of lung squamous cell carcinoma.

A genetic interaction between RAP1 and telomerase reveals an unanticipated role for RAP1 in telomere maintenance.

RAP1 is one of the components of shelterin, the capping complex at chromosome ends or telomeres, although its role in telomere length maintenance and protection has remained elusive. RAP1 also binds subtelomeric repeats and along chromosome arms, where it regulates gene expression and has been shown to function in metabolism control. Telomerase is the enzyme that elongates telomeres, and its deficiency causes a premature aging in humans and mice. We describe an unanticipated genetic interaction between RAP1 and telomerase. While RAP1 deficiency alone does not impact on mouse survival, mice lacking both RAP1 and telomerase show a progressively decreased survival with increasing mouse generations compared to telomerase single mutants. Telomere shortening is more pronounced in Rap1-/- Terc-/- doubly deficient mice than in the single-mutant Terc-/- counterparts, leading to an earlier onset of telomere-induced DNA damage and degenerative pathologies. Telomerase deficiency abolishes obesity and liver steatohepatitis provoked by RAP1 deficiency. Using genomewide ChIP sequencing, we find that progressive telomere shortening owing to telomerase deficiency leads to re-localization of RAP1 from telomeres and subtelomeric regions to extratelomeric sites in a genomewide manner. These findings suggest that although in the presence of sufficient telomere reserve RAP1 is not a key factor for telomere maintenance and protection, it plays a crucial role in the context of telomerase deficiency, thus in agreement with its evolutionary conservation as a telomere component from yeast to humans.

G6PD protects from oxidative damage and improves healthspan in mice.

Reactive oxygen species (ROS) are constantly generated by cells and ROS-derived damage contributes to ageing. Protection against oxidative damage largely relies on the reductive power of NAPDH, whose levels are mostly determined by the enzyme glucose-6-phosphate dehydrogenase (G6PD). Here, we report a transgenic mouse model with moderate overexpression of human G6PD under its endogenous promoter. Importantly, G6PD-Tg mice have higher levels of NADPH, lower levels of ROS-derived damage, and better protection from ageing-associated functional decline, including extended median lifespan in females. The G6PD transgene has no effect on tumour development, even after combining with various tumour-prone genetic alterations. We conclude that a modest increase in G6PD activity is beneficial for healthspan through increased NADPH levels and protection from the deleterious effects of ROS.

Mice with Pulmonary Fibrosis Driven by Telomere Dysfunction.

Idiopathic pulmonary fibrosis (IPF) is a degenerative disease of the lungs with an average survival post-diagnosis of 2-3 years. New therapeutic targets and treatments are necessary. Mutations in components of the telomere-maintenance enzyme telomerase or in proteins important for telomere protection are found in both familial and sporadic IPF cases. However, the lack of mouse models that faithfully recapitulate the human disease has hampered new advances. Here, we generate two independent mouse models that develop IPF owing to either critically short telomeres (telomerase-deficient mice) or severe telomere dysfunction in the absence of telomere shortening (mice with Trf1 deletion in type II alveolar cells). We show that both mouse models develop pulmonary fibrosis through induction of telomere damage, thus providing proof of principle of the causal role of DNA damage stemming from dysfunctional telomeres in IPF development and identifying telomeres as promising targets for new treatments.

Growth Arrest Specific 1 (Gas1) Gene Overexpression in Liver Reduces the In Vivo Progression of Murine Hepatocellular Carcinoma and Partially Restores Gene Expression Levels.

The prognosis of hepatocellular carcinoma patients is usually poor, the size of tumors being a limiting factor for surgical treatments. Present results suggest that the overexpression of Gas1 (growth arrest specific 1) gene reduces the size, proliferating activity and malignancy of liver tumors. Mice developing diethylnitrosamine-induced hepatocellular carcinoma were subjected to hydrodynamic gene delivery to overexpress Gas1 in liver. This treatment significantly (p < 0.05) reduced the number of large tumors, while the difference in the total number of lesions was not significant. Moreover, the number of carcinoma foci in the liver and the number of lung metastases were reduced. These results are related with the finding that overexpression of Gas1 in Hepa 1-6 cells arrests cell cycle before S phase, with a significant (p < 0.01) and concomitant reduction in the expression of cyclin E2 gene. In addition, a triangular analysis of microarray data shows that Gas1 overexpression restores the transcription levels of 150 genes whose expression was affected in the diethylnitrosamine-induced tumors, thirteen of which are involved in the hedgehog signaling pathway. Since the in vivo Gas1 gene delivery to livers of mice carrying hepatocellular carcinoma reduces the size and proliferating activity of tumors, partially restoring the transcriptional profile of the liver, the present study opens promising insights towards a therapeutic approach for hepatocellular carcinoma.

Therapeutic inhibition of TRF1 impairs the growth of p53-deficient K-RasG12V-induced lung cancer by induction of telomeric DNA damage.

Telomeres are considered anti-cancer targets, as telomere maintenance above a minimum length is necessary for cancer growth. Telomerase abrogation in cancer-prone mouse models, however, only decreased tumor growth after several mouse generations when telomeres reach a critically short length, and this effect was lost upon p53 mutation. Here, we address whether induction of telomere uncapping by inhibition of the TRF1 shelterin protein can effectively block cancer growth independently of telomere length. We show that genetic Trf1 ablation impairs the growth of p53-null K-Ras(G12V)-induced lung carcinomas and increases mouse survival independently of telomere length. This is accompanied by induction of telomeric DNA damage, apoptosis, decreased proliferation, and G2 arrest. Long-term whole-body Trf1 deletion in adult mice did not impact on mouse survival and viability, although some mice showed a moderately decreased cellularity in bone marrow and blood. Importantly, inhibition of TRF1 binding to telomeres by small molecules blocks the growth of already established lung carcinomas without affecting mouse survival or tissue function. Thus, induction of acute telomere uncapping emerges as a potential new therapeutic target for lung cancer.

Role of cannabinoid receptor CB2 in HER2 pro-oncogenic signaling in breast cancer.

BACKGROUND: Pharmacological activation of cannabinoid receptors elicits antitumoral responses in different cancer models. However, the biological role of these receptors in tumor physio-pathology is still unknown. METHODS: We analyzed CB2 cannabinoid receptor protein expression in two series of 166 and 483 breast tumor samples operated in the University Hospitals of Kiel, Tübingen, and Freiburg between 1997 and 2010 and CB2 mRNA expression in previously published DNA microarray datasets. The role of CB2 in oncogenesis was studied by generating a mouse line that expresses the human V-Erb-B2 Avian Erythroblastic Leukemia Viral Oncogene Homolog 2 (HER2) rat ortholog (neu) and lacks CB2 and by a variety of biochemical and cell biology approaches in human breast cancer cells in culture and in vivo, upon modulation of CB2 expression by si/shRNAs and overexpression plasmids. CB2-HER2 molecular interaction was studied by colocalization, coimmunoprecipitation, and proximity ligation assays. Statistical tests were two-sided. RESULTS: We show an association between elevated CB2 expression in HER2+ breast tumors and poor patient prognosis (decreased overall survival, hazard ratio [HR] = 0.29, 95% confidence interval [CI] = 0.09 to 0.71, P = .009) and higher probability to suffer local recurrence (HR = 0.09, 95% CI = 0.049 to 0.54, P = .003) and to develop distant metastases (HR = 0.33, 95% CI = 0.13 to 0.75, P = .009). We also demonstrate that genetic inactivation of CB2 impairs tumor generation and progression in MMTV-neu mice. Moreover, we show that HER2 upregulates CB2 expression by activating the transcription factor ELK1 via the ERK cascade and that an increased CB2 expression activates the HER2 pro-oncogenic signaling at the level of the tyrosine kinase c-SRC. Finally, we show HER2 and CB2 form heteromers in cancer cells. CONCLUSIONS: Our findings reveal an unprecedented role of CB2 as a pivotal regulator of HER2 pro-oncogenic signaling in breast cancer, and they suggest that CB2 may be a biomarker with prognostic value in these tumors.

Distinct p21 requirements for regulating normal and self-reactive T cells through IFN-γ production.

Self/non-self discrimination characterizes immunity and allows responses against pathogens but not self-antigens. Understanding the principles that govern this process is essential for designing autoimmunity treatments. p21 is thought to attenuate autoreactivity by limiting T cell expansion. Here, we provide direct evidence for a p21 role in controlling autoimmune T cell autoreactivity without affecting normal T cell responses. We studied C57BL/6, C57BL/6/lpr and MRL/lpr mice overexpressing p21 in T cells, and showed reduced autoreactivity and lymphadenopathy in C57BL/6/lpr, and reduced mortality in MRL/lpr mice. p21 inhibited effector/memory CD4(+) CD8(+) and CD4(-)CD8(-) lpr T cell accumulation without altering defective lpr apoptosis. This was mediated by a previously non-described p21 function in limiting T cell overactivation and overproduction of IFN-γ, a key lupus cytokine. p21 did not affect normal T cell responses, revealing differential p21 requirements for autoreactive and normal T cell activity regulation. The underlying concept of these findings suggests potential treatments for lupus and autoimmune lymphoproliferative syndrome, without compromising normal immunity.

TRIB3 suppresses tumorigenesis by controlling mTORC2/AKT/FOXO signaling.

In a recent article, we found that Tribbles pseudokinase 3 (TRIB3) plays a tumor suppressor role and that this effect relies on the dysregulation of the phosphorylation of v-akt murine thymoma viral oncogene homolog (AKT) by the mammalian target of rapamycin complex 2 (mTORC2 complex), and the subsequent hyperphosphorylation and inactivation of the transcription factor Forkhead box O3 (FOXO3).

Sox4 links tumor suppression to accelerated aging in mice by modulating stem cell activation.

Sox4 expression is restricted in mammals to embryonic structures and some adult tissues, such as lymphoid organs, pancreas, intestine, and skin. During embryogenesis, Sox4 regulates mesenchymal and neural progenitor survival, as well as lymphocyte and myeloid differentiation, and contributes to pancreas, bone, and heart development. Aberrant Sox4 expression is linked to malignant transformation and metastasis in several types of cancer. To understand the role of Sox4 in the adult organism, we first generated mice with reduced whole-body Sox4 expression. These mice display accelerated aging and reduced cancer incidence. To specifically address a role for Sox4 in adult stem cells, we conditionally deleted Sox4 (Sox4(cKO)) in stratified epithelia. Sox4(cKO) mice show increased skin stem cell quiescence and resistance to chemical carcinogenesis concomitantly with downregulation of cell cycle, DNA repair, and activated hair follicle stem cell pathways. Altogether, these findings highlight the importance of Sox4 in regulating adult tissue homeostasis and cancer.