RENCA Macrobeads Inhibit Tumor Cell Growth via EGFR Activation and Regulation of MEF2 Isoform Expression

Tumors are heterogeneous systems, whose growth is influenced by intrinsic properties of malignant cells, external systemic factors (i.e. immune, neural, endocrine, etc.), and the dynamic interactions between tumor cells and their microenvironment. Given the inherent complexity of cancers, combined with the continual evolution of tumors and the development of treatment resistance, a precision medicine approach may not provide an optimal clinical response. Exploring a new paradigm that focuses on regulating cancer as a system may not only control tumor progression but also address the extraordinary challenges of tumor heterogeneity and disease recurrence in order to improve clinical outcomes. As a group of discrete, growth-restricted tumor colonies that regulate their own growth and secrete a large number of tumor-inhibitory signals, RENCA macrobeads function as a biological-system, providing the opportunity for a systems-therapeutic approach to cancer management. Previous work has demonstrated that RENCA macrobeads restrict the growth of various cancer cells both in vitro as well as in preclinical and clinical studies; however, the molecular mechanism(s) of this inhibition is unknown. In this study, we demonstrated that factors secreted by RENCA macrobeads significantly altered the transcript levels of multiple MEF2 isoforms in targeted tumor cells. Suppression of various MEF2 isoforms markedly reduced the growth inhibitory effect of RENCA macrobeads and abrogated macrobead induced S-phase arrest. Importantly, we identified an essential role for the MEF2D isoform in mediating RENCA macrobead-induced inhibition. In addition, the cell-surface receptor, EGFR, was shown to be involved in the anti-proliferative response to RENCA macrobeads. Growth inhibition was more robust in cells overexpressing EGFR and was associated with cell accumulation in S-phase. In cell lines with reduced EGFR kinase activity or low-levels of cell-surface receptor, we demonstrated that RENCA macrobeads inhibited growth, although to a lesser degree and exhibited G2/M arrest, supporting the notion that factors secreted by RENCA macrobeads regulate multiple cell cycle checkpoints. Lastly, we identified three proteins in conditioned media of RENCA macrobeads (RTN4, TSP1, TIMP2) that partially contribute to growth regulation of external tumor cells with functional EGFR activity. Moreover, we identified a novel role for these proteins in modulating MEF2 activity and regulating MEF2 expression, particularly the MEF2D isoform. Overall, these studies support a mechanism by which RENCA macrobeads, at least partially, regulate tumor growth external to the macrobead. These findings could identify patients most likely to benefit from RENCA macrobead therapy

MEF2 plays a significant role in the tumor inhibitory mechanism of encapsulated RENCA cells via EGF receptor signaling in target tumor cells

Abstract Background Agarose encapsulated murine renal adenocarcinoma cells (RENCA macrobeads) are currently being investigated in clinical trials as a treatment for therapy-resistant metastatic colorectal cancer. We have previously demonstrated the capacity of RENCA macrobeads to produce diffusible substances that markedly inhibit the proliferation of epithelial-derived tumor cells outside the macrobead environment. This study examined the molecular mechanisms underlying the observed inhibition in targeted tumor cells exposed to RENCA macrobeads. Methods We evaluated changes in transcription factor responses, participating intracellular signaling pathways and the involvement of specific cellular receptors in targeted tumor cells exposed to RENCA macrobeads. Results Factors secreted by RENCA macrobeads significantly up-regulated the activity of the MEF2 transcription factor as well as altered the transcription of MEF2b and MEF2d isoforms in targeted tumor cells. Suppression of individual or multiple MEF2 isoforms in target tumor cells markedly reduced the growth inhibitory effects of RENCA macrobeads. Furthermore, these effects were linked to the activation of the EGF receptor as attenuation of EGFR resulted in a substantial reduction of the cancer cell growth-inhibitory effect. Conclusions Since interruption of the EGFR signaling cascade did not eliminate RENCA macrobead-induced growth control, our data suggests that RENCA macrobeads exert their full growth inhibitory effects through the simultaneous activation of multiple signaling pathways. In contrast to a precision medicine approach targeting single molecular abnormalities, the RENCA macrobead functions as a biological-systems therapy to re-establish regulation in a highly dysfunctional and dysregulated cancer system