Abstract
Epithelial to mesenchymal transition (EMT) has become one of the most exciting fields in cancer biology. While its role in cancer cell invasion, metastasis and drug resistance is well established, the molecular basis of EMT-induced immune escape remains unknown. We recently reported that EMT coordinately regulates target cell recognition and sensitivity to specific lysis. In addition to the well-characterized role for EMT in tumor phenotypic change including a tumor-initiating cell phenotype, we provided evidence indicating that EMT-induced tumor cell resistance to cytotoxic T-lymphocytes (CTLs) also correlates with autophagy induction. Silencing of BECN1 in target cells that have gone through the EMT restored CTL susceptibility to CTL-induced lysis. Although EMT may represent a critical target for the development of novel immunotherapy approaches, a more detailed understanding of the inter-relationship between EMT and autophagy and their reciprocal regulation will be a key determinant in the rational approach to future tumor immunotherapy design.
Keywords: epithelial mesenchymal transition, autophagy, cytotoxic T-lymphocytes, breast cancer
It is well established that the growth and spread of cancer depends as much on the host response to tumor as on the biological characteristics of the tumor itself. This interaction is at its most intimate and dynamic within the tumor microenvironment. In this regard, evidence has been accumulated clearly indicating that antitumor CTL induction is not sufficient because effector T cells are downregulated in situ. This is in part due to the plasticity and complexity of the tumor microenvironment. In this respect, there are increasing indications that the tumor stroma components play a crucial role in the control of the local immune response, and contain many overlapping mechanisms to maintain tumor functional disorder and evasion of antigen-specific CTLs.
In a recent report, using the breast MCF-7-derived tumor cells that have undergone EMT following overexpression of wild-type SNAI1/SNAIL or the constitutively activated (SNAI1-6SA) protein, or by the acquisition of TNFα resistance (2101 cells), we showed that EMT transcription factors are not the only way to induce an enhanced phenotypic plasticity resulting in breast cancer cell resistance to CTLs. We also show that the acquisition of resistance to TNF leads to EMT induction and the subsequent resistance to antigen-specific killer cells. It is worth noting that the acquisition of resistance to TNF and the high EMT score of TNF-resistant (2101) cells suggest the existence of a level of complexity in the EMT process in which multiple molecules act together to mediate EMT, rather than the master regulators acting on their own.
Consistent with the role of autophagy as a cell-protective mechanism, we investigated whether the activation of the EMT program in tumor cells is associated with the induction of autophagy. Our results showed that expression of SNAI1 in breast cancer cells induces an epithelial dedifferentiation program that coincides with a drastic change in cell morphology and the activation of autophagy flux. Interestingly, we found that BECN1 is upregulated in mesenchymal cells compared with epithelial cells. Although the molecular mechanism by which the EMT program affects the expression of BECN1 remained to be addressed, several lines of evidence indicate that this may be related to SNAI1- or EMT-dependent repression of a miRNA(s) involved in modulation of BECN1 expression. Indeed, it has been reported that MIR30A inhibits the expression of BECN1, and that the transcription factors SNAI1 and ZEB1 bound to E-boxes in the MIR34A/B/C promoters, thereby repressing MIR34A and MIR34B/C expression. While much remains to be learned mechanistically, this result extended the role of SNAI1 as a regulator of autophagy and paves the way to an interesting topic of research. Although targeting BECN1 in mesenchymal cells is sufficient to restore CTL-mediated tumor cell lysis, it has no effect on cell morphology and the expression of EMT markers. This finding suggests that autophagy is a downstream target of the EMT program in breast cancer cells. It is worth noting that our results are not in line with other recently published reports showing that autophagy promotes hepatocellular carcinoma cell invasion through activation of the EMT.
To identify transcriptional changes of other autophagy core machinery genes that are specific responses toward SNAI1 expression, a comparative differential gene expression analysis using cDNA microarrays was performed. We did not detect any modulation of ATG genes in SNAI1-overexpressing breast cancer cells, suggesting that SNAI1 or the EMT program induces autophagy independently of the modulation in the expression of ATG genes. Data mining of microarray results predicted, however, a functional link between an EMT marker and some autophagy inducers and repressors. These data strongly suggest that the EMT activates autophagy flux indirectly by regulating key autophagy repressor or inducer genes. In order to further understand the relationship between the EMT and tumor resistance to immune surveillance, ongoing studies are focusing on the analysis of the precise contribution of EMT transcription factors in autophagy regulation and how this homeostatic process is mechanistically linked to the maintenance of breast stem cell marker expression in tumor cells.
Our studies suggest EMT-induced autophagy is a novel mechanism by which tumor cells regulate CTL reactivity and impede their cytotoxic activity and further point to the complex relationship between the tumor system and the immune system. They also suggest that the selection of cells that have gone through the EMT, through autophagy-induced resistance, among epithelial tumor cells may originate not only from signals produced by the nontumor cells that constitute the tumor microenvironment, but also by the immune effectors infiltrating the tumors and their secreted cytokines (i.e., TNF, TGFβ1). This also suggests that the antigen-specific killer cells not only have antitumor function, but also paradoxically immunoedits tumors, facilitating tumor escape and progression. Although much remains to be explored in the EMT-MET (mesenchymal epithelial transition) cycle in controlling metastasis, the systematic determination of EMT score may improve the therapeutic management of breast carcinomas, and strategies to target cells that have gone through the EMT might hold promise for prevention or treatment.
Tumor stroma components are engaged in an active molecular crosstalk that has serious implications for immunological recognition of tumor in shaping the microenvironment. Accordingly, the various strategies aimed at the induction of antitumor cytotoxic responses should therefore consider the morphological changes described in this report as an antitumor mechanism of tumor escape, partly involved in resistance of tumor cells to cytotoxicity. Therefore, a better understanding of the crosstalk between cytotoxic T cells and tumor stroma-induced EMT as well as the molecular mechanisms that regulate EMT-induced resistance to cell death of cells that are committed to progress and invade, may open new perspectives for the development of novel treatment strategies to enhance the efficacy of cancer immunotherapy. Our results clearly suggest new insights into immunotherapeutic strategies by manipulating EMT and autophagy to avoid tumor immune evasion. However, preclinical work will be needed to test more specific autophagy inhibitors and block autophagy in tumors, as well as allow EMT targeting (Fig. 1).
Figure 1. The acquisition of an EMT phenotype in the context of microenvironmental stroma reactivity confers resistance to CTL-mediated lysis through autophagy induction.
Acknowledgments
This work was supported by funding from the French league against cancer, the “Association de Recherche sur le Cancer” (ARC) to S.C., and grant FC/2012/02 from “Fondation Cancer,” Luxembourg to B.J.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Footnotes
Previously published online: www.landesbioscience.com/journals/autophagy/article/24728