Colonic carcinogenesis involves the progressive dysregulation of homeostatic mechanisms that control growth. The epidermal growth factor (EGF) receptor (EGFR) regulates colonocyte growth and differentiation and is overexpressed in many human colon cancers. A requirement for EGFR in colonic premalignancy, however, has not been shown. In the current study, we used a specific EGFR antagonist, gefitinib, to investigate this role of the receptor in azoxymethane colonic premalignancy. The azoxymethane model shares many clinical, histologic, and molecular features of human colon cancer. Mice received azoxymethane i.p. (5 mg/kg/wk) or saline for 6 weeks. Animals were also gavaged with gefitinib (10 mg/kg body weight) or vehicle (DMSO) thrice weekly for 18 weeks, a dose schedule that inhibited normal receptor activation by exogenous EGF. Compared with control colonocytes [bromodeoxyuridine (BrdUrd), 2.2 F 1.2%], azoxymethane significantly increased proliferation (BrdUrd, 12.6 F 2.8%), whereas gefitinib inhibited this hyperproliferation (BrdUrd, 6.2 F 4.0%; <0.005). Azoxymethane significantly induced pro-transforming growth factor-A (6.4 F 1.3-fold) and increased phospho-(active) EGFR (5.9 F 1.1-fold), phospho-(active) ErbB2 (2.3 F 0.2-fold), and phospho-(active) extracellular signal-regulated kinase (3.3 F 0.4-fold) in premalignant colonocytes. Gefitinib inhibited activations of these kinases by >75% (P < 0.05). Gefitinib also significantly reduced the number of large aberrant crypt foci and decreased the incidence of colonic microadenomas from 75% to 33% (P < 0.05). Gefitinib concomitantly decreased cell cycle-regulating cyclin D1 and prostanoid biosynthetic enzyme cyclooxygenase-2 in microadenomas, suggesting that these regulators are key targets of EGFR in colonic carcinogenesis. These results show for the first time that EGFR signaling is required for early stages of colonic carcinogenesis. Our findings suggest, moreover, that inhibitors of EGFR might be useful in chemopreventive strategies in individuals at increased risk for colonic malignancies. [Cancer Res 2007;67(2):827-35]
Aberrant crypt foci (ACF) are collections of abnormal colonic crypts with heterogeneous molecular and pathologic characteristics. Large and dysplastic ACF are putative precursors of colon cancer with neoplastic risk related to increased proliferation. In this study, we examined the role of epidermal growth factor receptor (EGFR) signaling in regulating ACF proliferation. Using magnification chromoendoscopy, we collected large ACF with endoscopic features of dysplasia and separately biopsied adjacent mucosa. Transcript levels were measured by real-time PCR, proteins were assessed by Western blotting, and levels were expressed as fold changes of adjacent mucosa. K-ras and B-Raf mutations were assessed by PCR and Ras activation by the ratio Ras-GTP / (Ras-GTP + Ras-GDP). At the RNA level, 38% of ACF were hyperproliferative, with proliferating cell nuclear antigen (PCNA) mRNA z2-fold of adjacent mucosa. Hyperproliferative ACF had significantly increased mRNA levels of EGFR (6.0 F 1.7-fold), transforming growth factor-A (14.4 F 5.0-fold), heparin-binding EGF-like growth factor (4.5 F 1.4-fold), cyclin D1 (4.6 F 0.7-fold), and cyclooxygenase-2 (COX-2; 9.3 F 4.2-fold; P < 0.05). At the protein level, 46% of ACF were hyperproliferative (PCNA, 3.2 F 1.2-fold). In hyperproliferative ACF, 44% possessed significant increases in four EGFR signaling components: EGFR (9.5 F 1.3-fold), phosphoactive ErbB2 (2.6 F 0.4-fold), phosphoactive extracellular signal-regulated kinase (3.7 F 1.1-fold), and cyclin D1 (3.4 F 0.8-fold; P < 0.05). Ras was activated in 46% of ACF (3.2 F 0.4-fold; P < 0.05), but K-ras mutations were present in only 7% of ACF. In contrast to COX-2 mRNA, the protein was not increased in hyperproliferative ACF. In summary, we have shown that ACF with up-regulated PCNA possess increased EGFR signaling components that likely contribute to the enhanced proliferative state of
Purpose: Colonic carcinogenesis deranges growth-regulating epidermal growth factor receptors (EGFR). We previously showed that EGFR signals were up-regulated in human aberrant crypt foci (ACF), putative colon cancer precursors. The azoxymethane model of colon cancer recapitulates many aspects of human colonic tumors. Recent studies indicate that flat dysplastic ACF with increased h-catenin are tumor precursors in this model. We asked, therefore, if EGFR signals are required for flat dysplastic ACF development and cancer progression. Experimental Design: Rats received azoxymethane or saline, and standard chow or chow supplemented with gefitinib, an EGFR inhibitor, for 44 weeks. EGFR signals were quantified in normal colon, flat ACF, and tumors by computerized analysis of immunostains and Western blots. K-ras mutations were assessed by PCR and mRNA for egfr ligands by quantitative real-time PCR. Results: EGFR inhibition with gefitinib decreased the incidence of flat dysplastic ACF from 66% to 36% and tumors from 71% to 22% (P < 0.05). This inhibitor also reduced the overexpressions of cyclin D1 and Cox-2 in flat ACF. Furthermore, in flat ACF, EGFR blockade decreased the upregulation of c-Jun, FosB, phosphorylated active signal transducers and activators of transcription 3, and CCAAT/enhancer binding protein-h, potential regulators of cyclin D1 and Cox-2. In colonic tumors, EGFR blockade significantly decreased angiogenesis, proliferation, and progression while also increasing apoptosis (P < 0.05). Gefitinib also inhibited the activations of extracellular signal^regulated kinase, Src, and AKT pathways in tumors. Conclusions: We have shown for the first time that EGFR promotes the development of flat dysplastic ACF and the progression of malignant colonic tumors. Furthermore, we have mechanistically identified several transcription factors and their targets as EGFR effectors in colonic carcinogenesis.Colonic carcinogenesis is characterized by the accumulation of activating mutations in proto-oncogenes and inhibiting mutations in tumor suppressor genes. These mutations dysregulate pathways, including epidermal growth factor receptor (EGFR) signals that control cell growth, maturation, and cell death. Up-regulations of EGFRs and ligands have been described in many tumors, including colon cancers (1). Recently, we reported that EGFR signals were up-regulated in human aberrant crypt foci (ACF) identified in situ using image magnification chromoendoscopy (2). ACF are the earliest identifiable lesions in experimental colonic carcinogenesis and dysplastic ACF are believed to be precursors of colon cancer (3).EGFR (ErbB1) is a member of the ErbB family of receptor tyrosine kinases which also includes ErbB2, ErbB3, and ErbB4 (4). Ligand binding induces a conformational change, causing receptors to dimerize and activating the receptor's intrinsic tyrosine kinase. ErbB2 is unique in that it has no identified ligand, but is the preferred heterodimeric partner for other members. EGFR signals activate multiple pathways i...
Aging is a major risk factor in increased lung cancer incidence. While most research has focused on age-associated mutation accumulation to explain the late-life increase in cancer incidence, there are tissue environmental forces that both impede and promote cancer evolution. Just as organismal evolution is known to be driven by environmental changes, cellular (somatic) evolution in our bodies is similarly driven by changes in tissue environments. Environmental change promotes selection for new phenotypes that are adaptive to the new context. In our tissues, aging or insult-driven alterations in tissues drives selection for adaptive mutations, and some of these mutations can confer malignant phenotypes. Chronic, low-level inflammation has been associated with aging, termed inflammaging, yet how age-associated changes in lung tissue microenvironments contribute to increased lung cancer incidence has remained largely unknown. Since chronic inflammation has been shown to contribute to tumor development, we hypothesized that inflammaging contributes to increased oncogenic adaptation in the lung. Using either viral delivery of CRISPR constructs to mediate EML4-ALK translocations or ectopic expression of KRAS-G12D, we showed increased adenoma formation in old mice. Importantly, in the EML4-ALK model, we showed that the overexpression of alpha-1 antitrypsin (AAT) in old mice resulted in lower adenoma counts compared to their old wild type counterparts. Flow cytometric analysis of immune cells isolated from bronchoalveolar fluid of young and old mice showed an altered immune landscape, such as increased neutrophils, gamma delta T cells, and Foxp3+ regulatory T cells. Furthermore, analysis of the single-cell RNAseq data from Tabula Muris Consortium demonstrated increased exhaustion markers in the CD8+ T cells and regulatory T cells. Separately, Gene Set Enrichment Analysis (GSEA) of the differential gene expressions of lung epithelial cells isolated from young and old mice revealed enriched pathways related to immune activation and inflammatory response, and immune-suppression markers. Lastly, bulk RNA-seq from lungs of young, old, and old mice overexpressing AAT revealed increased immune cell exhaustion markers and that the overexpression of AAT partially reversed this increase. Finally, analysis of Genotype-Tissue Expression (GTEx) data comparing gene expressions in lungs of young and old humans similarly showed enriched pathways related to immune activation and increased T cell exhaustion markers in the elderly. In addition, using deconvolution methods CiberSort and xCell, we demonstrated altered innate and adaptive immune cell populations, for example, increased neutrophils and regulatory T cells, that are associated with advanced age, similar to aging mice. In conclusion, we showed that there is an exhausted immune microenvironment in aging lungs, that inflammation contributes to the increased tumor initiation, and that decreasing inflammation could decrease the lung tumor incidence by reactivating the immune system. Citation Format: Shi Biao Chia, Catherine Pham-Danis, Hannah Scarborough, Nathaniel Little, Etienne P. Danis, Andrew E. Goodspeed, Charles Dinarello, James DeGregori. Altered immune landscape in aging lungs contributes to malignant evolution [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr A026.
Why do we get cancer? Why is cancer highly associated with old age? Of course, aging is associated with the accumulation of more mutations, and some of these mutations can contribute to cancer phenotypes. But we now understand that carcinogenesis is much more complex than originally appreciated. In particular, there are tissue environmental forces that both impede and promote cancer evolution. Just as organismal evolution is known to be driven by environmental changes, cellular (somatic) evolution in our bodies is similarly driven by changes in tissue environments, whether caused by the normal process of aging, by lifestyle choices or by extrinsic exposures. Environmental change promotes selection for new phenotypes that are adaptive to the new context. In our tissues, aging or insult-driven alterations in tissues drives selection for adaptive mutations, and some of these mutations can confer malignant phenotypes. We have been using mouse models of cancer initiation, mathematical models of cellular evolution, and analyses of human tissue samples to better understand the evolutionary forces that control somatic cell evolution and thus cancer risk. We have shown that aging and inflammation dependent changes in tissue environments dramatically dictate whether cancer-causing mutations are advantageous to stem cells in our tissues, starting the cells down the path to cancer. Our studies have focused on cancer initiation within the hematopoietic system and the lung. These studies have also uncovered molecular explanations for mutation-driven adaptation to aged and inflammatory tissue environments. In all, these studies indicate that strategies to prevent or treat cancers will need to incorporate interventions that alter tissue microenvironments. While we largely cannot prevent mutation accumulation through our lives, we do have the ability to manipulate tissue environments so as to change the evolutionary trajectories of cells with cancer-causing mutations. Citation Format: Catherine Pham-Danis, Andrii Rozhok, Hannah Scarborough, Nathaniel Little, Curtis Henry, Travis Nemkov, Kirk Hansen, James DeGregori. In the light of evolution: Why do we get more cancers in old age? [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr IA13.
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