p19 Arf Suppresses Growth, Progression, and Metastasis of Hras-Driven Carcinomas through p53-Dependent and -Independent Pathways

Ectopic expression of oncogenes such as Ras induces expression of p19^Arf, which, in turn, activates p53 and growth arrest. Here, we used a multistage model of squamous cell carcinoma development to investigate the functional interactions between Ras, p19^Arf, and p53 during tumor progression in the mouse. Skin tumors were induced in wild-type, p19^Arf-deficient, and p53-deficient mice using the DMBA/TPA two-step protocol. Activating mutations in Hras were detected in all papillomas and carcinomas examined, regardless of genotype. Relative to wild-type mice, the growth rate of papillomas was greater in p19^Arf-deficient mice, and reduced in p53-deficient mice. Malignant conversion of papillomas to squamous cell carcinomas, as well as metastasis to lymph nodes and lungs, was markedly accelerated in both p19^Arf- and p53-deficient mice. Thus, p19^Arf inhibits the growth rate of tumors in a p53-independent manner. Through its regulation of p53, p19^Arf also suppresses malignant conversion and metastasis. p53 expression was upregulated in papillomas from wild-type but not p19^Arf-null mice, and p53 mutations were more frequently seen in wild-type than in p19^Arf-null carcinomas. This indicates that selection for p53 mutations is a direct result of signaling from the initiating oncogenic lesion, Hras, acting through p19^Arf.     

The p53R172H Mutant Does Not Enhance Hepatocellular Carcinoma Development and Progression

Hepatocellular carcinoma is a highly deadly malignancy, accounting for approximately 800,000 deaths worldwide every year. Mutation of the p53 tumor suppressor gene is a common genetic change in HCC, present in 30% of cases. p53^R175H (corresponding to p53^R172H in mice) is a hotspot for mutation that demonstrates “prometastatic” gain-of-function in other cancer models. Since the frequency of p53 mutation increases with tumor grade in HCC, we hypothesized that p53^R172H is a gain-of-function mutation in HCC that contributes to a decrease in tumor-free survival and an increase in metastasis. In an HCC mouse model, we found that p53^R172H/flox mice do not have decreased survival, increased tumor incidence, or increased metastasis, relative to p53^flox/flox littermates. Analysis of cell lines derived from both genotypes indicated that there are no differences in anchorage-independent growth and cell migration. However, shRNA-mediated knockdown of mutant p53 in p53^R172H-expressing HCC cell lines resulted in decreased cell migration and anchorage-independent growth. Thus, although p53 mutant-expressing cells and tumors do not have enhanced properties relative to their p53 null counterparts, p53^R172H-expressing HCC cells depend on this mutant for their transformation. p53 mutants have been previously shown to bind and inhibit the p53 family proteins p63 and p73. Interestingly, we find that the levels of p63 and p73 target genes are similar in p53 mutant and p53 null HCC cells. These data suggest that pathways regulated by these p53 family members are similarly impacted by p53^R172H in mutant expressing cells, and by alternate mechanisms in p53 null cells, resulting in equivalent phenotypes. Consistent with this, we find that p53 null HCC cell lines display lower levels of the TA isoforms of p63 and p73 and higher levels of ΔNp63. Taken together these data point to the importance of p63 and p73 in constraining HCC progression.     

p19Arf Suppresses Growth, Progression, and Metastasis of Hras-Driven Carcinomas through p53-Dependent and -Independent Pathways

Ectopic expression of oncogenes such as Ras induces expression of p19^Arf, which, in turn, activates p53 and growth arrest. Here, we used a multistage model of squamous cell carcinoma development to investigate the functional interactions between Ras, p19^Arf, and p53 during tumor progression in the mouse. Skin tumors were induced in wild-type, p19^Arf-deficient, and p53-deficient mice using the DMBA/TPA two-step protocol. Activating mutations in Hras were detected in all papillomas and carcinomas examined, regardless of genotype. Relative to wild-type mice, the growth rate of papillomas was greater in p19^Arf-deficient mice, and reduced in p53-deficient mice. Malignant conversion of papillomas to squamous cell carcinomas, as well as metastasis to lymph nodes and lungs, was markedly accelerated in both p19 ^Arf- and p53-deficient mice. Thus, p19^Arf inhibits the growth rate of tumors in a p53-independent manner. Through its regulation of p53, p19^Arf also suppresses malignant conversion and metastasis. p53 expression was upregulated in papillomas from wild-type but not p19^Arf-null mice, and p53 mutations were more frequently seen in wild-type than in p19^Arf-null carcinomas. This indicates that selection for p53 mutations is a direct result of signaling from the initiating oncogenic lesion, Hras, acting through p19^Arf.     

Mutant p53 gain of function in two mouse models of Li-Fraumeni syndrome

The p53 tumor suppressor gene is commonly altered in human tumors, predominantly through missense mutations that result in accumulation of mutant p53 protein. These mutations may confer dominant-negative or gain-of-function properties to p53. To ascertain the physiological effects of p53 point mutation, the structural mutant p53R172H and the contact mutant p53R270H (codons 175 and 273 in humans) were engineered into the endogenous p53 locus in mice. p53R270H/+ and p53R172H/+ mice are models of Li-Fraumeni Syndrome; they developed allele-specific tumor spectra distinct from p53+/- mice. In addition, p53R270H/- and p53R172H/- mice developed novel tumors compared to p53-/- mice, including a variety of carcinomas and more frequent endothelial tumors. Dominant effects that varied by allele and function were observed in primary cells derived from p53R270H/+ and p53R172H/+ mice. These results demonstrate that point mutant p53 alleles expressed under physiological control have enhanced oncogenic potential beyond the simple loss of p53 function.     

Inactivation and Inducible Oncogenic Mutation of p53 in Gene Targeted Pigs

Mutation of the tumor suppressor p53 plays a major role in human carcinogenesis. Here we describe gene-targeted porcine mesenchymal stem cells (MSCs) and live pigs carrying a latent TP53^R167H mutant allele, orthologous to oncogenic human mutant TP53^R175H and mouse Trp53^R172H, that can be activated by Cre recombination. MSCs carrying the latent TP53^R167H mutant allele were analyzed in vitro. Homozygous cells were p53 deficient, and on continued culture exhibited more rapid proliferation, anchorage independent growth, and resistance to the apoptosis-inducing chemotherapeutic drug doxorubicin, all characteristic of cellular transformation. Cre mediated recombination activated the latent TP53^R167H allele as predicted, and in homozygous cells expressed mutant p53-R167H protein at a level ten-fold greater than wild-type MSCs, consistent with the elevated levels found in human cancer cells. Gene targeted MSCs were used for nuclear transfer and fifteen viable piglets were produced carrying the latent TP53^R167H mutant allele in heterozygous form. These animals will allow study of p53 deficiency and expression of mutant p53-R167H to model human germline, or spontaneous somatic p53 mutation. This work represents the first inactivation and mutation of the gatekeeper tumor suppressor gene TP53 in a non-rodent mammal.     

Induction of invasive mouse skin carcinomas in transgenic mice with mutations in both H-ras and p53

Synergistic interaction between H-ras and p53 were systematically examined during skin tumorigenesis. Concurrent expression of an activated H-ras gene and a mutant p53 gene was accomplished by crossing p53(Val135/wt) mice with TG.AC mice. Topical application to wild-type mice with benzo(a)pyrene (BaP) alone produced approximately 26% skin tumor incidence, whereas BaP treatment of p53(wt/wt)Hras(TG.AC/wt), p53(Val135/wt)Hras(wt/wt), and p53(Val135/wt)Hras(TG.AC/wt) mice produced a 75%, 77%, and 100% incidence of skin tumors, respectively. An average of 0.33 tumor per mouse was observed in wild-type (p53(wt/wt)Hras(wt/wt)) mice, whereas approximately 1.54, 1.96, and 3.08 tumors per mouse were seen in BaP-treated p53(wt/wt)Hras(TG.AC/wt), p53(Val135/wt)Hras(wt/wt), and p53(Val135/wt)Hras(TG.AC/wt) mice, respectively. The effects on total tumor volume were even more striking with 7-, 48-, and 588-fold increases in tumor volume compared with wild-type (p53(wt/wt)Hras(wt/wt)) in p53(wt/wt)Hras(TG.AC/wt), p53(Val135/wt)Hras(wt/wt), and p53(Val135/wt)Hras(TG.AC/wt) mice, respectively. Histopathologically, all tumors from p53(wt/wt)Hras(wt/wt) mice were either papillomas or well-differentiated squamous cell carcinomas, whereas the tumors in p53(wt/wt)Hras(TG.AC/wt), p53(Val135/wt)Hras(wt/wt), and p53(Val135/wt)Hras(TG.AC/wt) mice were principally squamous cell carcinomas with varying degree of invasiveness. Particularly, tumors in p53(Val135/wt)Hras(TG.AC/wt) mice exhibited the most rapid growth and the extreme form of tumor invasion. Microarray analysis revealed that dominant-negative p53 (Val135) and activated H-ras affected several cellular processes involved in tumorigenesis possibly through its effects on apoptosis, cell cycle arrest, and Ras-mitogen-activated protein kinase pathways. The present study provides the first in vivo evidence that a germ line p53 mutation and activated H-ras act synergistically to profoundly enhance tumor progression.     

Carbon-Ion Beam Irradiation Kills X-Ray-Resistant p53-Null Cancer Cells by Inducing Mitotic Catastrophe

Background and Purpose

To understand the mechanisms involved in the strong killing effect of carbon-ion beam irradiation on cancer cells with TP53 tumor suppressor gene deficiencies.

Materials and Methods

DNA damage responses after carbon-ion beam or X-ray irradiation in isogenic HCT116 colorectal cancer cell lines with and without TP53 (p53^+/+ and p53^-/-, respectively) were analyzed as follows: cell survival by clonogenic assay, cell death modes by morphologic observation of DAPI-stained nuclei, DNA double-strand breaks (DSBs) by immunostaining of phosphorylated H2AX (γH2AX), and cell cycle by flow cytometry and immunostaining of Ser10-phosphorylated histone H3.

Results

The p53^-/- cells were more resistant than the p53^+/+ cells to X-ray irradiation, while the sensitivities of the p53^+/+ and p53^-/- cells to carbon-ion beam irradiation were comparable. X-ray and carbon-ion beam irradiations predominantly induced apoptosis of the p53^+/+ cells but not the p53^-/- cells. In the p53^-/- cells, carbon-ion beam irradiation, but not X-ray irradiation, markedly induced mitotic catastrophe that was associated with premature mitotic entry with harboring long-retained DSBs at 24 h post-irradiation.

Conclusions

Efficient induction of mitotic catastrophe in apoptosis-resistant p53-deficient cells implies a strong cancer cell-killing effect of carbon-ion beam irradiation that is independent of the p53 status, suggesting its biological advantage over X-ray treatment.     

Integrated miRNA and mRNA expression profiling of mouse mammary tumor models identifies miRNA signatures associated with mammary tumor lineage

Background  

MicroRNAs (miRNAs) are small, non-coding, endogenous RNAs involved in regulating gene expression and protein translation. miRNA expression profiling of human breast cancers has identified miRNAs related to the clinical diversity of the disease and potentially provides novel diagnostic and prognostic tools for breast cancer therapy. In order to further understand the associations between oncogenic drivers and miRNA expression in sub-types of breast cancer, we performed miRNA expression profiling on mammary tumors from eight well-characterized genetically engineered mouse (GEM) models of human breast cancer, including MMTV-H-Ras, -Her2/neu, -c-Myc, -PymT, -Wnt1 and C3(1)/SV40 T/t-antigen transgenic mice, BRCA1 ^fl/fl ;p53 ^+/-;MMTV-cre knock-out mice and the p53 ^fl/fl ;MMTV-cre transplant model.     

Wild-Type Hras Suppresses the Earliest Stages of Tumorigenesis in a Genetically Engineered Mouse Model of Pancreatic Cancer

Oncogenic, activating mutations in KRAS initiate pancreatic cancer. There are, however, two other Ras family members, Nras and Hras, which can be activated in the presence of oncogenic Kras. The role of these wild-type Ras proteins in cancer remains unclear, as their disruption has been shown to enhance or inhibit tumorigenesis depending upon the context. As pancreatic cancer is critically dependent upon Ras signaling, we tested and now report that loss of Hras increases tumor load and reduces survival in an oncogenic Kras-driven pancreatic adenocarcinoma mouse model. These effects were traced to the earliest stages of pancreatic cancer, suggesting that wild-type Hras may suppress tumor initiation. In normal cells, activated Ras can suppress proliferation through p53-dependent mechanisms. We find that the tumor suppressive effects of Hras are nullified in a homozygous mutant p53 background. As such, loss of wild-type Hras fosters the earliest stages of pancreatic cancer in a p53-dependent manner.     

Metformin Radiosensitizes p53-Deficient Colorectal Cancer Cells through Induction of G2/M Arrest and Inhibition of DNA Repair Proteins

The present study addressed whether the combination of metformin and ionizing radiation (IR) would show enhanced antitumor effects in radioresistant p53-deficient colorectal cancer cells, focusing on repair pathways for IR-induced DNA damage. Metformin caused a higher reduction in clonogenic survival as well as greater radiosensitization and inhibition of tumor growth of p53^-/- than of p53^+/+ colorectal cancer cells and xenografts. Metformin combined with IR induced accumulation of tumor cells in the G2/M phase and delayed the repair of IR-induced DNA damage. In addition, this combination significantly decreased levels of p53-related homologous recombination (HR) repair compared with IR alone, especially in p53^-/- colorectal cancer cells and tumors. In conclusion, metformin enhanced radiosensitivity by inducing G2/M arrest and reducing the expression of DNA repair proteins even in radioresistant HCT116 p53^-/- colorectal cancer cells and tumors. Our study provides a scientific rationale for the clinical use of metformin as a radiosensitizer in patients with p53-deficient colorectal tumors, which are often resistant to radiotherapy.     

Loss of PML cooperates with mutant p53 to drive more aggressive cancers in a gender-dependent manner

p53 mutations and downregulation of promyelocytic leukemia (PML) are common genetic alterations in human cancers. In healthy cells these two key tumor suppressors exist in a positive regulatory loop, promoting cell death and cellular senescence. However, the influence of their interplay on tumorigenesis has not been explored directly in vivo. The contribution of PML to mutant p53 driven cancer was evaluated in a mouse model harboring a p53 mutation (p53^{wild-type/R172H}) that recapitulates a frequent p53 mutation (p53^R175H) in human sporadic and Li-Fraumeni cancers. These mice with PML displayed perturbation of the hematopoietic compartment, manifested either as lymphoma or extramedullary hematopoiesis (EMH). EMH was associated with peripheral blood leucocytosis and macrocytic anemia, suggestive of myeloproliferative- myelodysplastic overlap. In contrast, a complete loss of PML from these mice resulted in a marked alteration in tumor profile. While the incidence of lymphomas was unaltered, EMH was not detected and the majority of mice succumbed to sarcomas. Further, males lacking PML exhibited a high incidence of soft tissue sarcomas and reduced survival, while females largely developed osteosarcomas, without impact on survival. Together, these findings demonstrate that PML is an important tumor suppressor dictating disease development in a pertinent mouse model of human cancer.

Key Points: (1) A mutant p53 allele disrupts hematopoiesis in mice, by promoting lymphomas and myeloproliferative / myelodysplastic overlap. (2) Coincidental p53 allele mutation and PML loss shifts the tumor profile toward sarcoma formation, which is paralleled in human leiomyosarcomas (indicated by immunohistochemistry; IHC).     

Conditionally Reprogrammed Normal and Transformed Mouse Mammary Epithelial Cells Display a Progenitor-Cell–Like Phenotype

Mammary epithelial (ME) cells cultured under conventional conditions senesce after several passages. Here, we demonstrate that mouse ME cells isolated from normal mammary glands or from mouse mammary tumor virus (MMTV)-Neu–induced mammary tumors, can be cultured indefinitely as conditionally reprogrammed cells (CRCs) on irradiated fibroblasts in the presence of the Rho kinase inhibitor Y-27632. Cell surface progenitor-associated markers are rapidly induced in normal mouse ME-CRCs relative to ME cells. However, the expression of certain mammary progenitor subpopulations, such as CD49f⁺ ESA⁺ CD44⁺, drops significantly in later passages. Nevertheless, mouse ME-CRCs grown in a three-dimensional extracellular matrix gave rise to mammary acinar structures. ME-CRCs isolated from MMTV-Neu transgenic mouse mammary tumors express high levels of HER2/neu, as well as tumor-initiating cell markers, such as CD44⁺, CD49f⁺, and ESA⁺ (EpCam). These patterns of expression are sustained in later CRC passages. Early and late passage ME-CRCs from MMTV-Neu tumors that were implanted in the mammary fat pads of syngeneic or nude mice developed vascular tumors that metastasized within 6 weeks of transplantation. Importantly, the histopathology of these tumors was indistinguishable from that of the parental tumors that develop in the MMTV-Neu mice. Application of the CRC system to mouse mammary epithelial cells provides an attractive model system to study the genetics and phenotype of normal and transformed mouse epithelium in a defined culture environment and in vivo transplant studies.     

Host cystathionine-γ lyase derived hydrogen sulfide protects against Pseudomonas aeruginosa sepsis

Hydrogen sulfide (H₂S) has recently been recognized as a novel gaseous transmitter with several anti-inflammatory properties. The role of host- derived H₂S in infections by Pseudomonas aeruginosa was investigated in clinical and mouse models. H₂S concentrations and survival was assessed in septic patients with lung infection. Animal experiments using a model of severe systemic multidrug-resistant P. aeruginosa infection were performed using mice with a constitutive knock-out of cystathionine-γ lyase (Cse) gene (Cse^-/-) and wild-type mice with a physiological expression (Cse^+/+). Experiments were repeated in mice after a) treatment with cyclophosphamide; b) bone marrow transplantation (BMT) from a Cse^+/+ donor; c) treatment with H₂S synthesis inhibitor aminooxyacetic acid (ΑΟΑΑ) or propargylglycine (PAG) and d) H₂S donor sodium thiosulfate (STS) or GYY3147. Bacterial loads and myeloperoxidase activity were measured in tissue samples. The expression of quorum sensing genes (QS) was determined in vivo and in vitro. Cytokine concentration was measured in serum and incubated splenocytes. Patients survivors at day 28 had significantly higher serum H₂S compared to non-survivors. A cut- off point of 5.3 μΜ discriminated survivors with sensitivity 92.3%. Mortality after 28 days was 30.9% and 93.7% in patients with H₂S higher and less than 5.3 μΜ (p = 7 x 10⁻⁶). In mice expression of Cse and application of STS afforded protection against infection with multidrug-resistant P. aeruginosa. Cyclophosphamide pretreatment eliminated the survival benefit of Cse^+/+ mice, whereas BMT increased the survival of Cse^-/- mice. Cse^-/- mice had increased pathogen loads compared to Cse^+/+ mice. Phagocytic activity of leukocytes from Cse^-/- mice was reduced but was restored after H₂S supplementation. An H₂S dependent down- regulation of quorum sensing genes of P.aeruginosa could be demonstrated in vivo and in vitro. Endogenous H₂S is a potential independent parameter correlating with the outcome of P. aeruginosa. H₂S provides resistance to infection by MDR bacterial pathogens.     

Involvement of p38α in the mitotic progression of p53-/- tetraploid cells

The tumor suppressor protein p53 plays a major role in preserving genomic stability. p53 suppresses a pathway leading from normal diploidy to neoplastic aneuploidy (via an intermediate metastable stage of tetraploidy) at two levels: first by preventing the generation/survival of tetraploid cells, and second by repressing their aberrant multipolar division. Here, we report the characterization of p53^-/- tetraploid cells, which - at difference with both their p53^-/- diploid and their p53^+/+ tetraploid counterparts - manifest a marked hyperphosporylation of the mitogen-activated protein kinase MAPK14 (best known as p38α) that is particularly strong during mitosis. In p53^-/- tetraploid cells, phosphorylated p38α accumulated at centrosomes during the metaphase and at midbodies during the telophase. Selective knockdown or pharmacological inhibition of p38α had a dramatic effect on p53^-/- (but not p53^+/+) tetraploids, causing the activation of the spindle assembly checkpoint, an arrest during the metaphase, a major increase in abnormal bipolar and monopolar mitoses, as well as an increment in the generation of multinuclear cells. We conclude that the mitotic progression of p53^-/- (but not p53^+/+) tetraploids heavily relies on p38α, revealing a novel function for this protein in the context of aneuploidizing cell divisions.     

p19Arf suppresses growth, progression, and metastasis of Hras-driven carcinomas through p53-dependent and -independent pathways

Ectopic expression of oncogenes such as Ras induces expression of p19(Arf), which, in turn, activates p53 and growth arrest. Here, we used a multistage model of squamous cell carcinoma development to investigate the functional interactions between Ras, p19(Arf), and p53 during tumor progression in the mouse. Skin tumors were induced in wild-type, p19(Arf)-deficient, and p53-deficient mice using the DMBA/TPA two-step protocol. Activating mutations in Hras were detected in all papillomas and carcinomas examined, regardless of genotype. Relative to wild-type mice, the growth rate of papillomas was greater in p19(Arf)-deficient mice, and reduced in p53-deficient mice. Malignant conversion of papillomas to squamous cell carcinomas, as well as metastasis to lymph nodes and lungs, was markedly accelerated in both p19 (Arf)- and p53-deficient mice. Thus, p19(Arf) inhibits the growth rate of tumors in a p53-independent manner. Through its regulation of p53, p19(Arf) also suppresses malignant conversion and metastasis. p53 expression was upregulated in papillomas from wild-type but not p19( Arf)-null mice, and p53 mutations were more frequently seen in wild-type than in p19( Arf)-null carcinomas. This indicates that selection for p53 mutations is a direct result of signaling from the initiating oncogenic lesion, Hras, acting through p19(Arf).     

Absence of p53-dependent apoptosis leads to UV radiation hypersensitivity,enhanced immunosuppression and cellular senescence

Genotoxic stress triggers the p53 tumor suppressor network to activate cellular responses that lead to cell cycle arrest, DNA repair, apoptosis or senescence. This network functions mainly through transactivation of different downstream targets, including cell cycle inhibitor p21, which is required for short-term cell cycle arrest or long-term cellular senescence, or proapoptotic genes such as p53 upregulated modulator of apoptosis (PUMA) and Noxa. However, the mechanism that switches from cell cycle arrest to apoptosis is still unknown. In this study, we found that mice harboring a hypomorphic mutant p53, R172P, a mutation that abrogates p53-mediated apoptosis while keeping cell cycle control mostly intact, are more susceptible to ultraviolet-B (UVB)-induced skin damage, inflammation, and immunosuppression than wild-type mice. p53^R172P embryonic fibroblasts (MEFs) are hypersensitive to UVB and prematurely senesce after UVB exposure, in stark contrast to wild-type MEFs, which undergo apoptosis. However, these mutant cells are able to repair UV-induced DNA lesions, indicating that the UV hypersensitive phenotype results from the subsequent damage response. Mutant MEFs show an induction of p53 and p21 after UVR, while wild-type MEFs additionally induce PUMA and Noxa. Importantly, p53^R172P MEFs failed to downregulate anti-apoptotic protein Bcl-2, which has been shown to play an important role in p53-dependent apoptosis. Taken together, these data demonstrate that in the absence of p53-mediated apoptosis, cells undergo cellular senescence to prevent genomic instability. Our results also indicate that p53-dependent apoptosis may play an active role in balancing cellular growth.     

Absence of p53-dependent apoptosis leads to UV radiation hypersensitivity,enhanced immunosuppression and cellular senescence

Genotoxic stress triggers the p53 tumor suppressor network to activate cellular responses that lead to cell cycle arrest, DNA repair, apoptosis or senescence. This network functions mainly through transactivation of different downstream targets, including cell cycle inhibitor p21, which is required for short-term cell cycle arrest or long-term cellular senescence, or proapoptotic genes such as p53 upregulated modulator of apoptosis (PUMA) and Noxa. However, the mechanism that switches from cell cycle arrest to apoptosis is still unknown. In this study, we found that mice harboring a hypomorphic mutant p53, R172P, a mutation that abrogates p53-mediated apoptosis while keeping cell cycle control mostly intact, are more susceptible to ultraviolet-B (UVB)-induced skin damage, inflammation and immunosuppression than wild-type mice. p53^R172P embryonic fibroblasts (MEFs) are hypersensitive to UVB and prematurely senesce after UVB exposure, in stark contrast to wild-type MEFs, which undergo apoptosis. However, these mutant cells are able to repair UV-induced DNA lesions, indicating that the UV-hypersensitive phenotype results from the subsequent damage response. Mutant MEFs show an induction of p53 and p21 after UVR, while wild-type MEFs additionally induce PUMA and Noxa. Importantly, p53^R172P MEFs failed to downregulate anti-apoptotic protein Bcl-2, which has been shown to play an important role in p53-dependent apoptosis. Taken together, these data demonstrate that in the absence of p53-mediated apoptosis, cells undergo cellular senescence to prevent genomic instability. Our results also indicate that p53-dependent apoptosis may play an active role in balancing cellular growth.Key words: UVB irradiation, p53, DNA damage, DNA damage responses, apoptosis, senescence     

Loss of p53 Attenuates the Contribution of IL-6 Deletion on Suppressed Tumor Progression and Extended Survival in Kras-Driven Murine Lung Cancer

Interleukin-6 (IL-6) is involved in lung cancer tumorigenesis, tumor progression, metastasis, and drug resistance. Previous studies show that blockade of IL-6 signaling can inhibit tumor growth and increase drug sensitivity in mouse models. Clinical trials in non-small cell lung cancer (NSCLC) reveal that IL-6 targeted therapy relieves NSCLC-related anemia and cachexia, although other clinical effects require further study. We crossed IL-6 ^-/- mice with Kras ^G12D mutant mice, which develop lung tumors after activation of mutant Kras ^G12D, to investigate whether IL-6 inhibition contributes to tumor progression and survival time in vivo. Kras ^G12D; IL-6 ^-/- mice exhibited increased tumorigenesis, but slower tumor growth and longer survival, than Kras ^G12D mice. Further, in order to investigate whether IL-6 deletion contributes to suppression of lung cancer metastasis, we generated Kras ^G12D; p53 ^flox/flox; IL-6 ^-/- mice, which developed lung cancer with a trend for reduced metastases and longer survival than Kras ^G12D; p53 ^flox/flox mice. Tumors from Kras ^G12D; IL-6 ^-/- mice showed increased expression of TNFα and decreased expression of CCL-19, CCL-20 and phosphorylated STAT3 (pSTAT3) than Kras ^G12D mice; however, these changes were not present between tumors from Kras ^G12D; p53 ^flox/flox; IL-6 ^-/- and Kras ^G12D; p53 ^flox/flox mice. Upregulation of pSTAT3 and phosphorylated AKT (pAKT) were observed in Kras ^G12D tumors with p53 deletion. Taken together, these results indicate that IL-6 deletion accelerates tumorigenesis but delays tumor progression and prolongs survival time in a Kras-driven mouse model of lung cancer. However, these effects can be attenuated by p53 deletion.