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.

Keywords: Cell therapy; EGFR; MEF2; Tumor inhibition.

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The authors declare that they have no competing interests.

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Figures

Fig. 1

Factors secreted by RENCA macrobeads alter the transcription factor activity and expression of MEF2. (a) RENCA cells, transiently transfected with pathway-focused transcription factor-responsive luciferase reporter constructs were exposed to naïve media or 5-day conditioned media from > 18 wk. RENCA macrobeads. Fold-change, calculated based on normalized luciferase activity of the conditioned media (CM) response relative to the naïve media response, is graphed in descending order. Columns, mean (n = 3); bar, SD. Dotted lines at 2 and 0.5 on the y-axis indicate the threshold for two-fold up- and down-regulation respectively. (b) MEF2 reporter activity in RENCA cells exposed to 5-day RENCA macrobead-conditioned media. Reporter activity in response to naïve media was used as a control. Fold-change was calculated for each sample relative to the naïve media sample. Each column represents the mean (n = 6–8) ± SD (primary axis). Mean inhibitory response of RENCA macrobeads on freely growing RENCA cells; red circles (n = 3) ± SD (secondary axis). (c) RENCA cells were co-cultured with > 18 wk. RENCA macrobeads for 5 days. RENCA cells cultured in naïve media served as a control. Total RNA was isolated and subjected to real-time PCR analysis for the expression of MEF2a, MEF2b, MEF2c and MEF2d. MEF2c was not detected in RENCA cells. Each column represents the mean (n = 3) ± SD. *p < 0.001, compared with naïve media

Fig. 2

MEF2 is integral in mediating the anti-proliferative effect of RENCA macrobeads. RENCA cells were transiently transfected with two-rounds of 1 μM non-targeting siRNA, MEF2a, MEF2b, MEF2d or combined MEF2a, b and d siRNAs (MEF2 pool) for 72 h followed by culture in naïve media or together with > 18 wk. RENCA macrobeads in a cell culture insert system for 5 days. Each column represents the mean absorbance value (n = 3) ± SD following neutral red staining, with growth inhibition calculated as the percent difference in absorbance between the indicated conditions. *p < 0.005 compared with naïve media

Fig. 3

RENCA macrobeads regulate the epidermal growth factor receptor in both murine and human cell lines. In-cell Western analysis of total EGFR, phosphorylated EGFR (pY1068) and overlay (normalized pY1068/EGFR) in (a) RENCA cells, and (b) DU145 cells following exposure to 5-day conditioned media (CM) from RENCA macrobeads at 1–2 weeks or > 18 weeks of age as indicated. Cells cultured in naïve media were used as a control. Each column represents the mean normalized intensity in arbitrary units (AU) (n = 6–8) ± SD. *p < 0.001, compared with naïve media

Fig. 4

Gefitinib limits basal and RENCA macrobead-mediated EGFR activity and contributes to an additive inhibitory effect in DU145 cells. In-cell Western analysis of total EGFR, phosphorylated EGFR (pY1068) and overlay (normalized pY1068/EGFR) in DU145 cells pre-treated with gefitinib (GF) or 0.01% DMSO for 2 h at the indicated concentrations followed by culture in (a) naïve media, or (b) 5-day conditioned media from > 18 wk. RENCA macrobeads. Each column represents the mean normalized intensity in arbitrary units (AU) (n = 6–8) ± SD. *p < 0.001, compared with vehicle control. (c) DU145 cells pre-treated with gefitinib or 0.01% DMSO were cultured with naïve media or together with > 18 wk. RENCA macrobeads in a cell culture insert system for 5 days. Cells cultured in naïve media were used as a control. Histogram represents percent survival calculated as the percent absorbance of co-cultured samples relative to naïve media samples. Each column represents the mean survival (n = 3) ± SD

Fig. 5

EGFR blockade results in partial attenuation of RENCA macrobead-mediated growth inhibition. (a) In-cell Western analysis of total EGFR, phosphorylated EGFR (pY1068) and overlay (normalized pY1068/EGFR) in DU145/GR cells pre-treated with 1 μM gefitinib (GF) or 0.01% DMSO followed by culture in naïve or 5-day conditioned media from > 18 wk. RENCA macrobeads. Each column represents the mean normalized intensity in arbitrary units (AU) (n = 6–8) ± SD. *p < 0.001, compared with vehicle control or conditioned media. (b) DU145/GR cells pre-treated with 1 μM gefitinib or 0.01% DMSO were cultured with naïve media or together with > 18 wk. RENCA macrobeads in a cell culture insert system for 5 days. Histogram represents percent survival calculated as the percent absorbance of co-cultured samples relative to naïve media samples. Each column represents the mean survival (n = 3) ± SD

Fig. 6

RENCA macrobeads modulate the expression of MEF2 isoforms through EGF receptor signaling. MEF2A, MEF2B, MEF2C and MEF2D expression was assessed by qRT-PCR on (a) DU145 or (b) DU145/GR cells cultured in naïve media or together with > 18 wk. RENCA macrobeads alone or pre-treated with 1 μM gefitinib followed by culture in media as described for 5 days. MEF2B expression was not detected in DU145 or DU145/GR cells. Each column represents the mean (n = 3) ± SD. *p < 0.005, compared with naïve media or gefitinib in naïve media