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.

IL-6 expression can be detected in lung tumors [25] and in 53% of lung cancer cell lines [26], and IL-6 pathways are activated in a human lung cancer stem cell line [27][28][29]. Functional assays suggest that IL-6 influences the ability of cancer cells to metastasize to distant sites [30,31] and that IL-6 promotes tumor growth in a paracrine fashion in vivo [4,26,32]. Therefore, it is perhaps not surprising that IL-6 knockdown, genetic ablation, or treatment with a neutralizing IL-6 antibody inhibits tumor growth in vivo [4,33]. Conversely, activation of IL-6 signaling contributes to resistance to epidermal growth factor receptor (EGFR) inhibitors in a mouse model of NSCLC [34,35], while blockade increases drug sensitivity in xenograft models [34].
An IL-6 monoclonal antibody therapy would be predicted to inhibit the inflammatory microenvironment in lung cancer. One such therapy, ALD518, has undergone preclinical and Phase I and II clinical trials. It appears to be well tolerated and ameliorates NSCLC-related anemia and cachexia [3], although the totality of clinical outcomes needs further study.
To assess the contribution of IL-6 signaling inhibition on tumor progression and survival time in vivo, we crossed IL-6 -/mice with mutant Kras G12D mice because IL-6 is a downstream effector of oncogenic Ras to promote tumorigenesis [4]. NSCLC is often diagnosed with metastasis and has a poor prognosis. The treatment and prevention of lung cancer metastases are major unmet needs [36]. Inactivating mutations in p53 are found in at least 50% of NSCLC cases [36], and Kras G12D activation accompanied by p53 deletion can cause lung tumor metastasis [37]. To study the function of IL-6 in metastasis, we also generated Kras G12D ; p53 flox/flox ; IL-6 -/-mice..

Materials and Methods
Mice IL-6 -/-mice were purchased from The Jackson Laboratory and maintained in sterile housing [38]. Conditional Lox-Stop-Lox Kras G12D (hereafter referred to as Kras G12D ) mice [39] and p53 flox/flox mice [40] were described previously. Kras G12D and Kras G12D ; lL-6 -/-mice were inoculated with 5 × 10 6 PFU of adenoviral Cre (adeno-Cre) by intranasal inhalation to activate oncogenic Kras G12D in the lungs. Kras G12D ; p53 flox/flox and Kras G12D ; p53 flox/flox ;IL-6 -/-mice were inoculated with 5 × 10 5 PFU of adeno-Cre. All experimental procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The protocol was approved by the Institutional Animal Care and Use Committee at Dana-Farber Cancer Institute (permit number 04-094). All surgeries were performed under Avertin anesthesia to minimize suffering. After euthanasia, organs, including heart, liver, spleen, kidney, stomach, intestine, spine, brain, breast, skin, and testis or ovary, were undergone gross inspection for metastases. Lung tumors adhered to the pleura were considered parietal pleural metastases. Suspected metastases were harvested and confirmed by histological features.

Proliferation analysis
At 20 weeks post-infection, mice were injected intraperitoneally with 10 μL of 10 mM BrdU in PBS per gram of body weight and euthanized after 2 hours. Whole lungs were harvested and processed as described above. At 400X magnification, all BrdU-positive tumor cell nuclei were counted within 3 microscope fields with the most BrdU-positive nuclei after review of the whole lung section. Four mice per genotype were analyzed. Same method was used to calculate Ki67labeled tumor cells on sections from mice 28 weeks postinfection with adeno-Cre.

Quantitative real-time PCR
mRNA was extracted from tumors of Kras G12D and Kras G12D ; lL-6 -/-mice 32 weeks post-infection and Kras G12D ; p53 flox/flox and Kras G12D ; p53 flox/flox ; IL-6 -/-mice around 16 weeks post-infection for analysis. 2μg total RNA was reverse transcribed to cDNA using SuperRT cDNA synthesis kit (Beijing CoWin Biosciences Co., Ltd. China). Real-time PCR was performed using the BioRad iQ5 Realtime PCR system and StepOnePlus Realtime PCR system (ABI) with Realtime PCR Master Mix containing SYBR Green (QPK-201 ,TOYOBO, Japan) and unique primers (Table S1). Three to four samples for each group were detected.Gene expression results were normalized to β-actin mRNA.

Statistical analysis
The Student's t-test was used to evaluate lesion number and number of BrdU or Ki67-positive cells. Fisher's exact test evaluated metastatic rate. Kaplan-Meier analysis evaluated survival time. Expression differences among four groups were analyzed by ANOVA. P<0.05 was considered statistically significant.

IL-6 deletion retards oncogenic Kras G12D -induced lung tumor progression
At 20 weeks post-infection, lung tumors in Kras G12D ; IL-6 -/mice were modestly smaller and less dense than those in Kras G12D mice ( Figure 1D and E). At 28 weeks post-infection, in comparison with Kras G12D mice, more lesions were observed in Kras G12D ; IL-6 -/-mice with the majority of lesions in early stages of tumor development ( Figure 1F). However, tumors 3-10 mm in diameter were observed in lungs of Kras G12D mice, while the majority of Kras G12D ; IL-6 -/-lung tumors were less than 1.5 mm ( Figure 1G-I). Further, although IL-6 signaling promotes skin tumor growth and angiogenesis in a paracrine fashion [4], we did not detect any difference between Kras G12D and Kras G12D ; IL-6 -/-mice after immunohistochemical staining with Endomucin, a microvessel density marker to measure angiogenesis index ( Figure 1J and K).

IL-6 deletion attenuates lung tumor proliferation
To determine whether tumor proliferation is affected by IL-6 deletion in vivo, we measured BrdU-labeling cells in lung tumors. Significantly fewer labeled nuclei were observed in lung sections from Kras G12D ; IL-6 -/-mice 20 weeks post-infection with adeno-Cre compared with those derived from control Kras G12D mice (Figure 2A-C). Similar results were observed from Ki67 staining in lung sections from Kras G12D and Kras G12D ; IL-6 -/-mice 28 weeks post-infection with adeno-Cre ( Figure S2). Expression of pERK, which acts downstream of Kras and is associated with cancer cell proliferation, was reduced in tumors from Kras G12D ; IL-6 -/-mice 20 weeks post-infection with adeno-Cre compared to Kras G12D mice ( Figure 2D and E). Caspase-3 staining revealed no differences in tumor cell apoptosis between Kras G12D and Kras G12D ; IL-6 -/-mice ( Figure 2F and G).
We also examined the nuclear localization of NF-κB subunit p65, which is important in cancer-related inflammation and malignant progression [44,45]. However, no significant localization change was observed among tumors from the four genotypes ( Figure S6). And no dramatic change was observed in β-catenin expression in nucleus ( Figure S6), which is related to lung cancer development [46]. Expression of pSTAT3, which is the main downstream target of IL-6, was reduced in some Kras G12D ; IL-6 -/-tumors ( Figure 5) but increased in p53-deleted tumors. These data indicated that IL-6 deletion altered tumor expression of some inflammatory cytokines, although these changes were weakened by p53 deletion.

Discussion
Previous studies have shown that carcinogen-induced tumorigenesis in IL-6 −/− mice is delayed by 1-2 weeks [4,47]; however, we found no difference in Kras G12D -induced tumor onset regardless of IL-6 deficiency. One possible explanation is that Kras G12D activation may induce lung tumorigenesis more robustly than other carcinogens. Some inflammatory cytokines are associated with tumor progression [42]. TNFα may act as a tumor promoter by regulating a cascade of cytokines, chemokines, adhesions, matrix metalloproteinases (MMPs) and pro-angiogenic activities [2,48]. In this study, IL-6 deletion in Kras G12D tumors upregulated TNFα expression. Elevated expression of TNFα may compensate for the loss of IL-6 and thus increase tumorigenesis. However, tumor progression is delayed in Kras G12D ; IL-6 -/-mice, consistent with previous results [4,47]. These data indicate that IL-6 is important for tumor progression in vivo and suggest that IL-6 inhibition may have biphasic stage-specific effects in lung cancer, enhancing tumorigenesis early while suppressing tumor progression later. Consequently, CCL-20 (or macrophage pro-inflammatory chemokine-3α, MIP-3α), a C-C motif chemokine, is overexpressed in pancreatic carcinoma cells and stimulates growth of tumor cells [49]. CCL-19 (or macrophage inflammatory protein-3 beta, MIP-3β), plays an important role in the migration of mature dendritic cells and T-cells [50]. Both dendritic cells and T-cells are double-edged swords in the tumor microenvironment, in addition to initiating potent anti-tumor immune responses, these cells may also stimulate cancerous cell growth and spreading [51,52]. Persistently activated or tyrosinephosphorylated STAT3 (pSTAT3) is found in 50% of lung adenocarcinomas [53,54]. pSTAT3 can enhance tumor proliferation and loss of pSTAT3 arrests growth of premalignant lesions, almost abrogating the development of advanced tumors [55]. In this study, IL-6 deletion in Kras G12D tumors resulted in downregulation of pSTAT3, CCL-19 and CCL-20.
pERK expression was reduced in Kras G12D ; IL-6 -/-tumors 20 weeks post-infection (Figure 2), but increased in most Kras G12D ; IL-6 -/-tumors 32 weeks post-infection ( Figure 5). These data suggest that early stage, tumor growth may be delayed by low expression of pERK, pSTAT3 and CCL-20. During later stages, tumor growth may be induced by upregulation of pERK and TNFα, although these mechanisms need further study. Table 2. Site and frequency of metastases from primary lung tumors.  Our data show that p53 deletion more dramatically affected Kras G12D -induced lung cancer than IL-6 deletion. To a large extent, p53 deletion attenuated the effects of IL-6 deletion on delayed tumor growth and prolonged survival. p53 deletion enhanced pSTAT3 expression ( Figure 5) and abrogated the change in CCL-20 expression in Kras G12D ; p53 flox/flox ; IL-6 -/tumors ( Figure 4). p53 deletion also increased expression of pAKT and total-AKT expression, which are associated with high proliferation, in Kras G12D ; p53 flox/flox and Kras G12D ; p53 flox/flox ; IL-6 -/-tumors( Figure 5). p53 deletion may attenuate the effects of IL-6 deletion through these pathways.

Sites of metastases
We observed a trend for reduced metastases with IL-6 deletion ( Table 2), although additional samples are required to confirm this result. Separately, we have observed dramatically increased IL-6 expression in primary and metastatic tumors from mice with high metastatic rates (unpublished data), similar to the report that IL-6 promotes cancer cells to metastasize to distant sites [30,31]. Furthermore, survival time of Kras G12D ; p53 flox/flox ; IL-6 -/-mice was significantly extended (P< 0.01) ( Table 1). These results indicate that IL-6 deletion may reduce lung cancer metastases and prolong survival time in vivo although p53 deletion dominantly impacts the evolution of Kras G12D lung cancer.
The involvement of inflammation in tumorigenesis, progression, and metastasis is widely accepted; however, whether IL-6-targeted therapies will prolong the survival time of lung cancer patients remains uncertain. Our results indicate anti-IL-6 therapies may have some success in clinical trials. For example, when NSCLC has not metastasized, IL-6 inhibition may prolong survival but increase the risk of further tumorigenesis; if metastasized, IL-6 inhibition may only moderately impact metastasis but may lengthen survival time. Further studies are needed to elucidate these possibilities. In summary, our results provide evidence that IL-6 deficiency promotes lung tumorigenesis, but suppresses tumor progression and elongates survival in vivo. However, these effects can be attenuated by p53 deletion.