A Novel Model for Squamous Cell Carcinoma of the Lung
Image of lungs from a kinase-dead IKKα knock-in (KA) mouse and a normal mouse. The KA mouse lung has multiple SCC tumors, which are indicated by arrows.
In the U.S. lung cancer remains the most deadly cancer type with less than one in five patients alive five years after diagnosis. The majority of lung cancer deaths are due to tobacco smoke, and the squamous cell carcinoma (SCC) subtype of lung cancer is strongly associated with smoking. Researchers have identified a number of mutations in lung SCC tumors but have failed to generate an animal model of lung SCC, which is critical for understanding the biology of the disease and for identifying novel therapeutic targets.
Yinling Hu, Ph.D., in CCR’s Laboratory of Experimental Immunology (LEI) and her colleagues have been studying the role the kinase IKKα plays in the development of SCC, predominantly in the skin. They then generated a lung SCC model in a kinase-dead IKKα knock-in (KA) mouse, in which lysine 44 is replaced with an alanine (K44A) in the ATP binding domain. The Hu laboratory and Qun Jiang, Ph.D., in the laboratory of Robert Wiltrout, Ph.D., teamed up and investigated the pathogenesis of lung SCCs.
At birth the KA mice appeared normal but, by 3 months, had severe skin lesions and systemic inflammation and began to die around 6 to 10 months. The researchers also identified spontaneous lung tumors in the KA mice between the ages of 4 and 10 months. Compared to normal mice, KA mice expressed lower IKKα levels in the lung, and the level dramatically decreased in mice four or more months old. Thus, the reductions in IKKα corresponded with lung tumor development.
To better study the importance of IKKα in lung tumor formation, the researchers generated two additional mouse models: the L-KA model, which re-expressed normal IKKα in the skin of KA mice, and the K-KA model, which re-expressed normal IKKα in the skin as well as the lungs of KA mice. The L-KA mice displayed no skin lesions, but all 50 mice examined by the investigators developed lung tumors. K-KA mice, however, lived longer lives, and none of the 30 mice examined developed lung tumors even after 12 months, suggesting that IKKα expression could prevent lung tumor growth.
Studying the L-KA mice further, the researchers noted that the mice’s lungs became heavier with age and that tumors formed when most of the mice were 4 to 6 months old, indicating that tumor development associated with increased lung size. The tumors the L-KA mice developed had characteristics of SCCs, including keratin pearls, squamous cell morphology, and expression of keratin 5, p63, and Ki67, which are typical of human lung SCCs, suggesting that these tumors are bone fide SCCs.
The investigators then looked for molecular changes in the lungs of the L-KA mice. Despite a lack of Ras activating mutations in mouse lung SCC, they found increases in stem cell regulators, such as Oct3/4 and Nanog, decreases in tumor suppressors, including p53, and activated pro-growth and -survival signaling proteins. The scientists also observed elevated expression of Trim29 and an N-terminal truncated version of p63 (ΔNp63), which have been used as diagnostic markers of human lung SCC, in the mice’s lungs that was further enhanced in lung SCCs. When the researchers investigated the molecular alterations in human lung SCC tumors, they noted similar changes in IKKα and Trim29 protein levels.
To understand how IKKα might regulate Trim29 and ΔNp63 expression, the researchers performed chromatin immunoprecipitation in normal mouse embryonic fibroblasts (MEFs). IKKα bound to the promoters of both the Trim29 and p63 genes. IKKα binding was associated with high levels of histone 3 lysine 27 trimethylation (H3K27me3), a transcription inhibiting modification, and low levels of histone 3 lysine 4 trimethylation (H3K4me3), a modification associated with increased transcription.
In KA MEFs and an L-KA SCC cell line, however, the researchers observed increased H3K4me3 and reduced H3K27me3 as well as higher levels of Trim29 and ΔNp63 mRNA. When the investigators re-expressed functional IKKα in these cells, H3K4me3 decreased and H3K27me3 increased at the Trim29 and p63 promoters. Expression of both genes also decreased. Additionally, reintroducing IKKα blocked proliferation of the L-KA SCC cells. These results suggest that IKKα controls the expression of Trim29 and ΔNp63 epigenetically and can regulate cell proliferation. The researchers found that IKKα uses a similar mechanism to regulate Trim29 and p63 in human cells.
Since inflammation can play a role in tumor development, the investigators examined the lungs of L-KA mice for markers of inflammation. They found significantly increased numbers of macrophages in the mice’ lungs as well as increased expression of a number of cytokines and chemokines, including tumor necrosis factor-α. Interestingly, the scientists also found increased macrophage infiltration into human lung SCC tumors.
To test whether inflammation was critical to lung SCC development, the researchers treated L-KA mice with liposomes containing clodronate, a drug that causes macrophage apoptosis. After three months of treatment, the investigators observed reduced lung weights and found no lung SCC tumors in the six treated mice. Macrophage depletion also reduced expression of Trim29, ΔNp63, cell cycle regulators, cytokines and chemokines, and markers of DNA damage. The researchers concluded that the increased number of macrophages enhanced lung inflammation and tumor formation in mice with low IKKα and their loss inhibited these processes.
The investigators took these results a step further by examining the role of bone marrow-derived macrophages on lung SCC formation. They irradiated wild type or L-KA mice to deplete their bone marrow then injected the mice with bone marrow derived from other wild type or L-KA mice and monitored lung SCC formation. All L-KA mice that received L-KA bone marrow developed lung SCCs while wild type mice that received wild type or L-KA bone marrow remained tumor free. Intriguingly, six out of seven L-KA mice that received wild type bone marrow also were free of lung tumors. The researchers suggested that the tumor in the seventh animal may have developed before the bone marrow transplant took place. Increased macrophages were also observed in human lung SCC and their adjacent lung tissues to SCC.
These data demonstrate that mice with reduced IKKα expression and increased pulmonary inflammation spontaneously generate lung SCCs that recapitulate the disease observed in the human lung and should be a useful tool for exploring new methods of preventing, detecting, and treating lung cancer in patients.Summary Posted: 04/2013
Xiao Z, Jiang Q, Willette-Brown J, Xi S, Zhu F, Burkett S, Back T, Song NY, Datla M, Sun Z, Goldszmid R, Lin F, Cohoon T, Pike K, Wu X, Schrump DS, Wong KK, Young HA, Trinchieri G, Wiltrout RH, and Hu Y. The Pivotal Role of IKKα in the Development of Spontaneous Lung Squamous Cell Carcinomas. Cancer Cell. April 15, 2013 PubMed Link