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Tumors are composed of cancer cells but also a larger number of diverse stromal cells in the tumor microenvironment. Stromal cells provide essential supports to tumor pathophysiology but the distinct characteristics of their signaling networks are not usually considered in developing drugs to target tumors. This oversight potentially confounds proof-of-concept studies and increases drug development risks. Here, we show in established murine and human models of breast cancer how differential regulation of Akt by the small GTPase RhoB in cancer cells or stromal endothelial cells determines their dormancy versus outgrowth when angiogenesis becomes critical. In cancer cells in vitro or in vivo, RhoB functions as a tumor suppressor that restricts EGF receptor (EGFR) cell surface occupancy as well as Akt signaling. However, after activation of the angiogenic switch, RhoB functions as a tumor promoter by sustaining endothelial Akt signaling, growth, and survival of stromal endothelial cells that mediate tumor neoangiogenesis. Altogether, the positive impact of RhoB on angiogenesis and progression supercedes its negative impact in cancer cells themselves. Our findings elucidate the dominant positive role of RhoB in cancer. More generally, they illustrate how differential gene function effects on signaling pathways in the tumor stromal component can complicate the challenge of developing therapeutics to target cancer pathophysiology. Cancer Res; 73(1); 50–61. ©2012 AACR.

The transcription factor, CCAAT/enhancer binding protein alpha (C/EBPalpha) is important in the terminal differentiation of granulocytes, hepatocytes, and adipocytes, and recurrent mutations of C/EBPalpha were described in acute myeloid leukemia. In the lung, C/EBPalpha is expressed in bronchial cells and type II pneumocytes. Abnormal proliferation of the latter cell type was reported in C/EBPalpha knockout mice. We determined the expression of C/EBPalpha by Northern blot analysis in 30 lung cancer cell lines and found significant down-regulation in 24 cell lines. Immunohistochemical study of primary tumor specimens showed undetectable or low expression of C/EBPalpha in 23 of 53 specimens. Its expression was more frequently down-regulated in adenocarcinoma and poorly differentiated cancer specimens than in squamous cell cancers. A higher frequency of reduced expression was found in more advanced stages. To investigate the consequences of C/EBPalpha expression in lung cancer cells, we stably transfected two cell lines that do not express the gene (Calu1 and H358) with a plasmid allowing for induction of C/EBPalpha protein expression. Induction of C/EBPalpha led to significant growth reduction attributable to proliferation arrest, morphological changes characteristic of differentiation, and apoptosis. These results suggest that C/EBPalpha is down-regulated in a large proportion of lung cancers and that it has growth-inhibitory properties in airway epithelial cells. Genetic analysis of the C/EBPalpha gene is in progress to fully evaluate its role as a novel tumor suppressor in lung cancer.

C/EBPα gene expression is frequently lost in non-small cell lung cancer, suggesting it acts as a tumor suppressor. Here, we generated an inducible lung-specific mouse model of C/EBPα deletion (C/EBPαLung-Δ mice) that develops lung adenocarcinomas. We observed that C/EBPα excision results in upregulation of Bmi-1, and that tumor initiation strictly depends on Bmi-1 gene dosage, as C/EBPαLung-Δ mice carrying only one functional Bmi-1 allele escape tumorigenesis. Accordingly, C/EBPα negative human adenocarcinomas show elevated Bmi-1 expression, in line with our observation that C/EBPα acts as tumor suppressor in lung cells by directly inhibiting Bmi-1 transcription. Furthermore, pharmacological inhibition of Bmi-1 impairs the ability of C/EBPα null adenocarcinoma cells to form tumors in xenografts. Overall, we have identified Bmi-1 as a critical therapeutic target in those patients carrying abnormal C/EBPα function. Citation Format: Elena Levantini, Daniela S. Basseres, Wen Cai Zhang, Robert S. Welner, Meritxell Alberich-Jorda’, Kol Jia Yong, Bhavin M. Thakkar, Junyan Zhang, Chiara Battelli, Christopher J. Hetherington, Min Ye, Karen O'Brien, Maria Cristina Magli, Marie Loh, Min En Nga, Yin Huei Pang, Alain C. Borczuk, Lyuba Varticovski, Olivier Kocher, Pu Zhang, Ross A. Soo, Bing Lim, Balazs Halmos, Daniel G. Tenen. C/EBPα acts as tumor suppressor in lung cancer by inhibiting the proto-oncogene Bmi-1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-46. doi:10.1158/1538-7445.AM2013-LB-46