ABSTRACT
In colorectal cancer (CRC), disease-related death is closely linked to tumor aggressiveness and metastasis. Gene expression profiling of patient tumors has suggested that a more mesenchymal phenotype, present in about one-fourth of all patients, is associated with increased aggressiveness. Accordingly, the mesenchymal transcription factor Slug/SNAI2 has been associated with decreased disease-free survival. To decipher the basis for the Slug-mediated phenotype, we conducted RNAseq experiments with a panel of HT-29 CRC cells expressing different levels of Slug, both in vitro and in tumor models. The results show that osteopontin, a secreted pleotropic protein involved in multiple steps of colorectal cancer progression, was highly upregulated by Slug in vitro, as well as in vivo. We further show that Slug is a direct regulator of osteopontin at the promoter level. The levels of secreted osteopontin were correlated with Slug expression, thereby linking the tumor phenotype to a biomarker available by liquid biopsies. The results also suggest that osteopontin neutralization may attenuate at least some of the Slug-mediated functions.
Subject(s)
Colorectal Neoplasms , Transcription Factors , Cell Line, Tumor , Colorectal Neoplasms/pathology , Humans , Osteopontin/genetics , Osteopontin/metabolism , Snail Family Transcription Factors/genetics , Snail Family Transcription Factors/metabolism , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Purpose: There is extensive cross-talk between VEGF- and EGFR-pathway signaling in colorectal cancer. However, combinations of VEGF- and EGFR-targeted monoclonal antibodies (mAb) show disappointing activity, in particular for patients with mutant RAS Previous results show that tyrosine kinase inhibitors (TKI) can be active in colorectal cancer models resistant to mAbs. This prompted us to examine whether the activity of bevacizumab can be increased by combination with erlotinib.Experimental Design: The antitumor activity of bevacizumab, erlotinib, and their combination was determined in colorectal cancer models with different RAS status and bevacizumab sensitivity. EGFR/VEGF pathway activation was characterized by immunohistochemistry, Western blot, and ELISA assays. The influence of cetuximab and erlotinib on EGF-mediated migration and the EGFR-EGF ligand feedback loop was established in colorectal cancer cell lines with different RAS status.Results: The addition of erlotinib increased bevacizumab activity in all models independent of RAS status. Bevacizumab exposure was accompanied by marked EGFR activation in tumor cells as well as in tumor-associated endothelial cells (TECs) and resulted in strong accumulation of intracellular EGFR, which could be attenuated by erlotinib. In cellular models, erlotinib was able to attenuate EGF-mediated functions in all cell lines independent of RAS status while cetuximab only showed activity in RAS wild-type cells.Conclusions: These results should provide a molecular framework to better understand the increased activity of the bevacizumab-erlotinib combination, compared with bevacizumab alone, in the GERCOR DREAM phase III clinical trial. Differential activity of mAbs and TKIs targeting the same signaling pathway is likely applicable for other tumor types. Clin Cancer Res; 24(11); 2548-58. ©2018 AACR.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Biomarkers, Tumor , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , ras Proteins/genetics , Animals , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Bevacizumab/administration & dosage , Cell Line, Tumor , Colorectal Neoplasms/pathology , Disease Models, Animal , Drug Synergism , Erlotinib Hydrochloride/administration & dosage , Humans , Mice , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/metabolism , Prognosis , Proto-Oncogene Proteins B-raf/genetics , Signal Transduction , Treatment Outcome , Xenograft Model Antitumor Assays , ras Proteins/metabolismABSTRACT
Most solid tumors contain a subfraction of cells with stem/progenitor cell features. Stem cells are naturally chemoresistant suggesting that chronic chemotherapeutic stress may select for cells with increased "stemness". We carried out a comprehensive molecular and functional analysis of six independently selected colorectal cancer (CRC) cell lines with acquired resistance to three different chemotherapeutic agents derived from two distinct parental cell lines. Chronic drug exposure resulted in complex alterations of stem cell markers that could be classified into three categories: 1) one cell line, HT-29/5-FU, showed increased "stemness" and WNT-signaling, 2) three cell lines showed decreased expression of stem cell markers, decreased aldehyde dehydrogenase activity, attenuated WNT-signaling and lost the capacity to form colonospheres and 3) two cell lines displayed prominent expression of ABC transporters with a heterogeneous response for stem cell markers. While WNT-signaling could be attenuated in the HT-29/5-FU cells by the WNT-signaling inhibitors ICG-001 and PKF-118, this was not accompanied by any selective growth inhibitory effect suggesting that the cytotoxic activity of these compounds is not directly linked to WNT-signaling inhibition. We conclude that classical WNT-signaling inhibitors have toxic off-target activities that need to be addressed for clinical development.
