Metastasis is a major cause of mortality and morbidity in breast cancer patients [1, 2]. The lung is one of the most common and deadliest sites of metastasis, especially for the most aggressive TNBC subtype [12, 30]. Despite advancements in current multimodality therapy options such as chemotherapy and radiotherapy, the median survival of breast cancer patients with lung metastases is approximately 11 months [12]. Several studies have shown that orchestrated interactions between disseminated cancer cell and metastatic microenvironment play a critical role in survival of cancer cells and formation of metastatic lesions [31–33]. Therefore, investigating the relationship between disseminated cancer cells and metastatic microenvironment will help in designing the most effective therapy options to treat or prevent lung metastasis. In this study we demonstrate that lung-derived soluble factors secreted in the presence of primary TNBC tumor play a critical role in influencing stemness/plasticity and metastatic behavior of TNBC cells.
Metastasis is a highly complex and an inefficient process as only a small percentage of disseminated cancer cells survive and grow at the secondary site following extravasation [7, 8]. Survival and growth of disseminated tumor cells mainly depends on cell-intrinsic (stemness/plasticity) and cell-extrinsic (favourable microenvironment at the secondary site) factors and the interaction between the two [34]. The preferential metastasis of TNBC to the lung suggests that lung provides a favourable nice for survival of these breast cancer cells. Even though breast cancer cells are successful in surviving and growing in the primary site, they often fail to grow and colonize distant organs [35]. This suggests that breast cancer cells with specific characteristics may have a better chance of survival and growth at metastatic sites. Our previous work has demonstrated that stem-like breast cancer cells, defined as cells with high ALDH activity and CD44 expression, displayed enhanced metastatic potential [15] and preferential metastasis to the lung [16].
In this current study, we observed that the lung microenvironment favoured growth and colonization of breast cancer cells with stem-like ALDHhiCD44+ characteristics as compared to non stem-like ALDHloCD44− breast cancer cells. We observed that, even though the whole cell population of TN breast cancer cells modestly colonized the lung, the ALDHhiCD44+ enriched subset of most aggressive SUM159 and MDA-MB-231 TNBC cells, displaying enhanced lung colonization and progression from single cell state to macrometastases by 21 days in the ex vivo pulmonary metastasis assay. The ability of stem-like cells to progress from single cell to macromateastases suggests that stemness/plasticity may be a key component for survival and metastatic colonization especially in the lung. Previously Pein et al showed that disseminated breast cancer cells with stem-like feature can induce pre-metastatic changes in lung fibroblasts to favor their survival and colonization [36]. In this study it is worth noting that in the ex vivo PuMA model, growth and colonization of stem-like ALDHhiCD44+ breast cancer occurred in the absence of pre-metastatic niche. This suggests that stem-like ALDHhiCD44+ cells may have possibly remodelled the lung microenvironment for survival and colonization after arrival in the lung. In addition, we also observed that even though the non stem-like breast cancer cells survived, they remain as single cells through in the ex vivo PuMA model. This suggests that a pre-metastatic niche may be required for colonization of breast cancer cells that lacking stem-like characteristics. It can be speculated that these non stem like single cells may remain dormant and upon acquisition of favourable conditions they could acquire stem-like phenotype and progress to macrometastasis. Several studies have demonstrated the stemness/plasticity often hinders effective therapy response through development of resistance to therapy [37, 38]. Therefore, elucidating the mechanism that confers stemness/plasticity would be beneficial in better treatment outcomes in metastatic patients. Taken together our results suggests that stem-like cell intrinsic factors that confer survival ability in an unfavourable microenvironment is very critical for successful metastasis. However, the mere presence of breast cancer cells with stem-like features does not always contribute to successful metastasis, suggesting that in addition to cell-intrinsic high ALDH activity and expression of CD44 in breast cancer cells, a supportive microenvironment is critical for successful metastatic process. growth and metastatic colonization breast cancer cells.
Stephen Paget’s “Seed and Soil” hypothesis proposes that a favourable microenvironment (soil) is critical for survival and growth of breast cancer cells (seed) at distant sites [10]. Interaction between disseminated tumor cells and the microenvironment at distant site is critical for metastasis [34]. Our previous work showed that presence of a primary TNBC breast tumor preferentially ‘primes’ the lung microenvironment thereby changes the secretion profiles of lung-derived soluble factors [17]. The primed lung microenvironment might be ideal in inducing stemness/plasticity in non stem-like breast cancer cells. In this study, we found that breast cancer cells exposed to LCM from TNBC-tumor bearing mice significantly increased acquisition of a stem-like phenotype and significantly decreased expression of a potential tumor suppressor, Dach1. Dach1 is a transcription factor involved in regulating different function during development [39]. In addition, it is known to suppress tumor cell growth [40–42], migration/invasion [24] stemness [26, 43] and metastasis [24]. We also observed that decreased expression of DACH1 in TNBC breast cancer patients was associated with poor overall survival and progression free survival and that inhibition of DACH1 significantly increased acquisition of a stem-like ALDHhiCD44+ phenotype in TNBC cells. Dach1 is known to negatively regulate stemness and inhibits expression of stem cell markers KLF4 and CD44, and epithelial to mesenchymal transition marker Vimentin [26]. We found that exposure of tbLCM or loss of function of DACH1 in breast cancer cells significantly increased expression of KLF4, CD44 and Vimentin, indicating that one or more secreted factors in the LCM derived from TNBC tumor-bearing mice influences stemness/plasticity in breast cancer cells.
To this end, we found that previously identified [16] lung-derived soluble factor FGF2 was important for metastatic colonization of TN breast cancer cells. FGF2 is known to play a key role in different cellular processes such as development [29], maintenance of normal stem cells [44, 45], cellular proliferation [46, 47], and angiogenesis [48]. It is also known to regulate cancer stem cell function [49, 50]. In this study we found that TNBC cells exposed to recombinant FGF2 resulted in significant increase in acquisition of stem-like ALDHhiCD44+ phenotype however the increase was moderate as compared to that observed in presence of the tbLCM. Furthermore, when we quantified the levels of FGF2 in tbLCM, to our surprise, we did not observe any significant difference in the levels of soluble FGF2 in lung conditioned media from tumor bearing (tbLCM) mice as compared to tumor naïve mice. This suggested that TNBC cells in the lung may rely on other lung-derived soluble in addition to FGF2 in influencing their stemness/plasticity.
Activation of FGF2 signaling results in perturbation of multiple signaling pathways and genes related to different cellular processes [29]. FGF2 secreted by osteogenic cells in the bone microenvironment induces stemness/plasticity in ER+ breast cancer cells by suppressing ER expression [51]. Interestingly, DACH1 is also known to suppress ERα in breast cancer cells [52]. This suggests a possible link between FGF2 and DACH1 in regulating different cellular processes. Interestingly, during skeletal development FGF2 upregulated DACH1 expression [28]. In preosteoblast cells, Dach1 played a key role in inhibiting FGF2 induced RANKL gene expression [53]. However, in glioma, Dach1 has been shown to transcriptionally suppress FGF2 expression [27]. This suggests a time and context dependent role of FGF2-Dach1 signaling axis in regulating different cellular function. In our current study, we found that TNBC cells treated with recombinant FGF2 protein significantly downregulated DACH1 expression. In addition, we also observed increase in expression of KLF4, CD44 and Vimentin expression which mimicked the loss of function of DACH1. Taken together these results suggested a link between FGF2 and Dach1 in influencing stemness/plasticity in breast cancer cells.
In conclusion, our study has demonstrated that the lung-derived soluble factors, particularly those secreted in the presence of primary TNBC tumor, play an important role in influencing stemness/plasticity and metastatic behaviour of breast cancer cells. Furthermore, we show that the FGF2-Dach1 signaling axis supports acquisition of stem-like ALDHhiCD44+ phenotype that is mostly favoured by the lung microenvironment for growth and metastatic colonization of the lung (Fig. 6). This pre-clinical work lays the foundation for future evaluation of FGF2 as a potential novel therapeutic target for treatment or prevention of breast cancer metastasis to the lung.