Chemoresistant Cancer Stem Cells Undergo Gene Changes That Drive Tumor Recurrence

Abstract Despite the efficacy of chemotherapy, resistant cells are thought to contribute to tumor recurrence. In vitro models of recurrence must mimic the complexity and heterogeneity of in vivo tumors and provide the longevity needed to capture tumor dormancy following chemotherapy. We have previously described a 3D in vitro model that simulates in vivo tumors containing cancer stem cell niches. Encapsulation of murine renal adenocarcinoma (RENCA) cells in a double layer of agarose that form spherical macrobeads allows for the development of 3D tumor colonies comprised of tumor-initiating and tumor-amplifying cells within the inner agarose matrix. In the current study, we used the RENCA macrobead in combination with chemotherapy to evaluate genetic regulation that allows tumor dormancy and drives recurrence. RENCA macrobeads treated with docetaxel (5 μg/ml) undergo a significant loss of cell mass and a period of latency followed by tumor recurrence. RNA isolated from docetaxel-resistant cells (Wk.6 post-treatment), recurrent tumors (Wk.20 post-treatment) and paired vehicle controls were characterized using whole-transcriptome profiling (GeneChip® WT Pico Kit with Transciptome Analysis Console; Affymetrix). At Wk.6 post-treatment, genes associated with a stem cell phenotype (Car12, Arrb1, Nos2, Aldh1l2) and members of the ABC1 family associated with multidrug resistance were enriched in docetaxel-resistant cells. Genes regulating the canonical Wnt signaling pathway, specifically LRP family members, were significantly up regulated. Pathway analysis revealed dysregulated focal adhesion-PI3K-Akt-mTor-signaling, including genes modulating integrin adhesion (Itga5, Itga7), ECM receptor interaction (Lama5, Lamb2, Lamc1) and actin cytoskeletal remodeling (Vcl). Supporting the quiescent nature of the docetaxel-resistant population, genes implicated in DNA replication (Dbf4, Cdc6, Mcm5), transcriptional initiation (Taf13, Gtf2h1, Polr2i) and tumor progression (Mmp3, Mmp13) were significantly down regulated. At Wk.20 post-treatment, up regulation of matrix metalloproteinases (Mmp13) and partial retention of stem cell (Nos2, Car12) and drug resistant (Abcb1) genes supports the evolution of regenerated tumors to a metastatic phenotype. Genes involved in fatty acid β-oxidation (Acsl5, Lipf), oxidative phosphorylation (ND3, ND6) and the electron transport chain (COX2) were down regulated in concert with up regulation of Me2 suggesting reduced flux through the TCA cycle and use of aerobic glycolysis in rapidly growing cells. This study further supports the RENCA macrobead as a model system to evaluate the biology of chemotherapy-resistant cells and tumor recurrence in a 3D microenvironment. These data are in line with the notion that chemotherapy-resistant cells exhibit a period of quiescence with a stem cell-like gene profile and experience an intrinsic transformation towards an invasive phenotype that drives tumor recurrence. Citation Format: Prithy C. Martis, Atira Dudley, Melissa A. Laramore, Hunter L. Gazda, Michael P. Markey, Barry H. Smith, Lawrence S. Gazda. Chemoresistant cancer stem cells undergo gene changes that drive tumor recurrence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3891. doi:10.1158/1538-7445.AM2017-3891