Breast cancer is a highly heterogeneous cancer type which possesses diverse phenotypic and morphologic features. Therefore, treatment strategies may vary for each breast cancer type. For example, hormone therapies are not helpful for Triple-Negative Breast Cancers or using anti-HER2 drugs is not to benefit for Luminal A type breast cancer which is tend be HER2− while this treatment strategy is used for HER2+ Luminal B. Also, inappropriate treatment application may cause various side effects or drug resistence development. So, accurate diagnosis and treatment is vital importance for breast cancer treatment such as other cancer types and development of novel therapeutic approaches or anti-cancer agents is needed and important for fighting aganist cancer.
Autophagy plays a survival role for normal cells because of being a mechanism which maintains energy homeostasis and is a source of nutriation and an elimination mechanism of damaged organelles and proteins. However, autophagy plays also important roles in carcinogenesis. In a tumor microenvironment, which contains stress conditions such as hypoxia, oxidative stress and nutrient deprivation, autophagy provides survival conditions by maintaining biomolecules and energy for cancer cells and causes adaptation of cancer cell to tumor microenvironment, therefore autophagy plays role as a tumor promoting oncogenic mechanism [40,41]. But, on the other hand, autophagy has a tumor suppressor role because of maintaining genomic stability, elimination of reactive oxygen species (ROS), degradation of oncogenic proteins and induction of immunresponse. Bidirectional role of autophagy in tumorigenesis depend on type and stage of cancer . Thus, relationship between autophagy and cancer still has been controversial. Among the programmed cell death mechanisms, espacially apoptosis has been widely studied in cancer biology. The first studies suggesting that apoptosis blocks tumor progression by elimination of malignant cells were performed in 1970s . The mechanisms which cause dysregulation of apoptosis and promote tumor progression can be summarized as disruption of the balance between pro-apoptotic and anti-apoptotic proteins, distortion of death receptor signals and reduction of caspase function . Cancer cells generally tend to evade from apoptosis and thus, they may gain capability of escape from immun system surveillence. In short, it is known that programmed cell death mechanisms are the first barriers preventing proliferation and survival of cancer cells. Therefore, various cancer treatment strategies targeting programmed cell death mechanisms have been developed and used until now. The type of programmed cell death which is induced by anti-cancer agent is depend on multiple factors such as type of cancer cell, dose of drugs, tumor microenvironment and type of cellular damage. So, to understand differences between programmed cell death mechanisms may contribute development of novel anti-cancer agents and treatment approaches.
In our previous study, we investigated cellular effects of the DC on lung cancer cells and revealed that the DC induced autophagic cell death in an apoptosis-independent manner in NSCLC cells . In this study, we aimed to investigate the biological effects of the DC in breast cancer according to metastatic features of cancer cells. We performed our experiments by using two different breast cancer cell lines, MDA-MB-231 and MCF-7 because of being cell culture models of different subtypes of breast cancer. MCF-7 cell line is estrogen receptor, progesterone receptor and HER2 positive, while MDA-MB-231 is negative. So, Luminal A breast cancer subtype MCF-7 is more appropriate cell line model for hormone therapy studies while triple-negative breast cancer subtype MDA-MB-231 cell line is generally used for chemotherapy and radiotherapy studies. MCF-7 cell line is a non-metastatic, but MDA-MB-231 is a highly metastatic cell line because of overexpression of EMT markers (Comşa et al., 2015; Subik et al., 2010).
Firstly, we examined the anti-proliferative effects of the DC on both breast cancer cell lines and determined that the DC suppressed proliferation of both cell lines. Depending on our previous study which showed that the DC induced autophagic cell death in NSCLC cell lines, we thought that the DC may induce programmed cell death mechanisms in breast cancer cell lines and we firstly evaluated protein expression levels of LC3BII for autophagy determination. We observed that autophagy was induced in MDA-MB-231 cell line during 48 hours, but it was only induced for the first hour and then it was reduced gradually during 48 hour-treatment in MCF-7 cell line (Fig. 3). This result showed that the DC induces a non-autophagic cell death mechanism in MCF-7 cell line unlike MDA-MB-231 cells. Depending on this result, we had two question; which cell death mechanism was induced by the DC in MCF-7 cell line and why these two breast cancer cell lines chose different cell death mechanisms? Therefore, we performed TUNEL assay for both the DC-treated cell lines and we obtained more signals in MCF-7 as compared to MDA-MB-231, indicating that MCF-7 underwent apoptosis process (Fig. 4a). To validate these results, we also evaluated protein expression levels of anti-apoptotic BCL-2 and pro-apoptotic BAX. The results which showed that the DC induced apoptosis in MCF-7 but not MDA-MB-231 cell line verified TUNEL assay results (Fig. 4b,c). Also, the DC reduced the invasive capability of metastatic MDA-MB-231 cells (Fig. 5). Upon this results, we wanted to elucidate the mechanisms which cause selection of different cell death pathways of breast cancer cell lines. Therefore, we hypothesized that the different cellular and genetic characteristics between the two cell lines may be responsible for these results. As is known, MCF-7 cell line is p53-mutant while MDA-MB-231 is p53-wild type. Tumor suppressor p53 is a key regulator which play important role in cell cyle arrest, maintaining cellular genomic integrity and controlling cell growth, senescence, differentiation, and apoptosis . p53-mediated growth inhibition depends on induction of an inhibitor of cyclin-dependent kinases p21 and p21 provides a functional link between p53 and cell cycle control [48,49]. Therefore, we evaluated protein expression levels of p53, p21 and another cell cycle and cell death regulator c-Myc. The results showed that p53 expression levels had not any significant change, p21 expression was induced relatively in both the DC-treated cell lines. But interestingly, c-Myc expression was induced significantly in MDA-MB-231, while it was downregulated in MCF-7 upon the DC treatment (Fig. 6). Next, to determine whether autophagy has a effect on the DC-induced c-Myc inhibition, we treated MCF-7 and MDA-MB-231 with Torin1 and chloroquine, are an inducer and a blocker of autophagy process, respectively. Chloroquine is a widely used autophagy inhibitor in the last stage of process by increasing lysosomal pH and blocking lysosome-autophagosome fusion . Torin1 prevents phosphorylation of downstream targets of mTORC1 and so induces autophagy . When we treated MDA-MB-231 cell line with the DC under autophagy-blocked conditions by chloroquine, we observed that c-Myc expression was downregulated significantly whereas c-Myc expression was upregulated upon only the DC treatment under normal conditions (Fig. 7b). Also, we induced autophagy with Torin1 in MCF-7 cell line and under autophagy-induced conditions the DC treatment could not repress c-Myc expression (Fig. 7a). According to these results, the DC downregulates c-Myc following induction of apoptosis in MCF-7 cells while it upregulates c-Myc following induction of autophagy in MDA-MB-231 cells. However, this is a recoverable mechanism via regulation of autophagy in both cell lines and we thought that there is a mutual interaction between autophagy and c-Myc. Because, MCF-7 has low expression level of c-Myc as compared to MDA-MB-231 [52,53]. In many studies, it was reported the interaction between autophagy and c-Myc (Z. Chen et al., 2019; Toh et al., 2013). Also, it was showed that Apigenin combined wih Gefitinib blocks autophagy by inducing apoptosis through inhibition of c-Myc . Recent studies showed that c-Myc overexpression promotes EMT by downregulating E-cadherin expression in breast cancer (Cho et al., 2010; Gao et al., 2019). EMT is a process that cells lose their epithelial phenotype and acquire mesenchymal features and it was reported that there is a crosstalk between autophagy and EMT in various cancer types in recent studies (H. T. Chen et al., 2019; Gugnoni et al., 2016). As previously mentioned, MCF-7 is a non-metastatic cell line and has a epithelial character while MDA-MB-231 has mesencymal phentype and highly metastatic capacity. Depending on this difference, we thought that metastatic features and expression levels of EMT markers of cell lines may determine the response to the DC, hence the cell death mechanism type. Therefore, we performed plasmid transfection for overexpression of Twist in MCF-7 cell line. Because low endogenous Twist expression is observed in MCF-7 as compared to MDA-MB-231 cell line and Twist upregulation may cause induction of EMT, invasion and migration of breast cancer cells . As a result of our analysis, we observed overexpression of c-Myc in exogenous Twist expressing MCF-7 cells treated the DC similar to MDA-MB-231 cell line (Fig. 8).
In recent studies, it was reported that cancer cells may induce autophagic cell death mechanism as a response to anti-cancer agents [66–68]. But, it is still unclear that clinic effects of autophagy on cancer. Therefore, as target mechanisms, selection of the distinctive mechanisms between normal cells and cancer cells is may be an useful approach for development of anti-cancer drugs. In this study, we determined that the DC induces autophagic cell death in metastatic breast cancer but it induces apoptotic cell death non-metastatic breast cancer. This different death pathway selections of different cancer cell lines may occur Twist/c-Myc axis, depending on metastatic features of breast cancer cells.
In conclusion, taken together with our previous study, the DC has anti-proliferative effects on lung and breast cancer and causes cell death. But determination of cell death mechanism type is depend on cancer cell type, even sub-type. Therefore, further studies is needed to illumination of detailed mechanisms of cellular effects of the DC and distinctive mechanisms between several cancer types. We hope that our findings may provide a novel perspective for interactions of autophagy, apoptosis and EMT pathways and may contribute to further analysis for development of novel anti-cancer therapies.