Principles of stem cell biology and cancer: future applications and therapeutics. Edited by T. Regad, T. J. Sayers and R. C. Rees. John Wiley & Sons (2015)
Part II. Cancer stem cells
Tumours produce a wide range of chemokines and cytokines, which function as ligands for MSC surface receptors (Dwyer et al., 2007). These cytokines and their corresponding receptors include SDF-1/CXCR4, SCbF/c-Kit, HGF/c-Met, VEGF/VEGF receptor, MCP/CCR2 and HMGB1/RAGE (Imitola et al., 2004; Schmidt et al., 2005; Son et al., 2006). In addition, MSCs produce a number of angiogenic growth factors that promote the growth of primary and metastatic tumours by inducing the formation of new blood vessels (De Luca et al., 2011). The tumour cells also secrete inflammatory cytokines and growth factors, such as IL-6, IL-8, neurotrophin-3, TGF-β, IL-1β, TNF-α, PDGF and EGF, which facilitate MSC migration towards tumour sites (Figure 7.1) (Nakamizo et al., 2005; Motaln et al., 2010). The MSCs are able to differentiate into stromal fibroblasts, which also interact with and influence the tumour cells through paracrine signals and various soluble factors (Erez et al., 2010). The crosstalk between bone marrow-derived MSCs and breast CSCs leads to an increase in the stem cell population (ALDH1+) in SUM159 cells, involving the IL-6 and CXCL7 loop, generated as a result of the interaction between two cell types (Korkaya et al., 2011b). Co-culture of tumour cells with MSCs also increases the percentage of tumour cells expressing the breast CSC markers CD24CD44+ . The expansion of the CSC population can be observed by the addition of MSC-derived conditioned medium, without any cell – cell contact, which shows that effect is due to the soluble factors (IL-6, CXCL7) released by MSCs in the conditioned medium.
CSCs contribute to tumour cell invasion and metastasis via the cascade of autocrine and paracrine signals from the tumour microenvironment. Well-known stem cell regulatory pathways are frequently dysregulated in tumour cells, including the Notch, Hh, Wnt, PI3K, NF-kB and Jak/STAT pathways (Korkaya et al., 2011a). In addition to mutational alterations, the tumour microenvironment also plays a vital role in the epigenetic activation of these developmental pathways during breast carcinogenesis. The tumour microenvironment contains MSCs and other cells that generate extrinsic factors such as Sonic hedgehog (Shh), Wnt, bone morphogenic proteins (BMPs), fibroblast growth factors (FGFs) and Notch, regulating the stem cell fate (Ivanova et al., 2002). When the equilibrium between these cellular signalling pathways is disrupted, dysregulated self-renewal may play a role in tumour development. It has been shown that tumour cells, as well as multiple cellular elements in the microenvironment, co-evolve during the process of carcinogenesis. Bidirectional paracrine signals coordinately regulate tumourigenic cell populations, including CSCs (Iliopoulos et al., 2009). Karnoub et al. (2007) previously demonstrated that paracrine signals produced by stromal cells play important roles in promoting breast cancer metastasis. MSCs within the tumour-associated stroma are critical determinants of cancer cell behaviour and phenotype. MSCs and derived cell types create a CSC niche to enable tumour progression via release of PGE2 and cytokines. Weinberg and colleagues reported that carcinoma cell-derived IL-1 induces prostaglandin E2 (PGE2 ) secretion by MSCs. The resulting PGE2 operates in an autocrine manner, cooperating with paracrine IL-1 signalling to induce expression of cytokines by the MSCs. The PGE2 and the cytokines then proceed to act in a paracrine fashion on the carcinoma cells to induce activation of β-catenin signalling and formation of CSCs (Li et al., 2012). CXCR4 and CXCL12 play an important role in the entry of breast cancer cells into the bone marrow, as well as in attaining breast cancer cell quiescence (Moharita et al., 2006). Inhibition of CXCR4, one of the signalling receptors for CXCL12, effectively inhibits both primary tumour growth and metastasis (Duda et al., 2011).
Figure 7.1. Crosstalk between MSCs and breast cancer cells causes migration of EMT CSCs and subsequent colonization at distant metastatic sites by MET CSCs.
Gene expression profiling has uncovered the transcription factor SOX4, which shows upregulated activity during TGF-β-induced EMT in normal and cancerous breast epithelial cells. SOX4 activation is important in EMT-induced tumour growth (Tiwari et al., 2013) and metastasis in both in vitro and in vivo models. MSCs are triggered by the expression of different micro-RNAs (miRs), including miR-335, known to regulate cellular interactions between the MSCs and breast tumours. The variable expression of miR-335 influences the activation of MSCs and the metastasis of breast cancer cells (Hass and Otte, 2012); miR-335 exerts its effect on more than 62 genes, including transcription factor SOX4 and the extracellular matrix component tenascin C, leading to enhancement of metastasis. Several studies have shown that IL-6, a proinflammatory cytokine, directly regulates the self-renewal of breast cancer cells through the activation of stat3, in a process mediated by the IL-6 receptor/GP130 complex (Sansone et al., 2007; Korkaya et al., 2012). During inflammation, IL-6-mediated Stat3 signalling selectively induces a protumourigenic microenvironment (Yu et al., 2009). Stat3 activation in turn leads to transcriptional activation of NF-kB in inflammatory cells, which secrete additional IL-6 and IL-8, acting on tumour cells. Thus, these cytokines generate a positive feedback loop between stromal cells and tumour cells, which further stimulates the CSC components by accelerating metastasis and therapeutic resistance. In addition, IL-6 has been shown to be a key component of a positive feedback loop involved in the expansion of breast CSCs through the bone marrow-derived MSC microenvironment (Korkaya et al., 2011b).
Notch signalling in bone marrow has been reported to act to maintain a pool of mesenchymal progenitors (Hilton et al., 2008). A recent study suggests that IL-6-meditated Jagged1-Notch promotes breast cancer bone metastasis, demonstrating that Jagged1 promotes tumour growth by stimulating release of IL-6 from osteoblasts and directly activating osteoclast differentiation (Sethi et al., 2011). The IL-8 secreted by MSCs activates multiple intracellular signalling pathways by binding its receptors, CXCR1 and CXCR2 (Korkaya et al., 2011a). Reports show that the aggressive behaviour and poor prognosis in patients with cancer are frequently associated with elevated serum IL-8 levels (Benoy et al., 2004; Yao et al., 2007). Increased expression of the IL-8 receptor, CXCR1, on breast CSCs has previously been reported (Ginestier et al., 2010). Recombinant IL-8 increases breast CSC self-renewal and tumour growth while blockade of this receptor in mouse xenografts reduces tumor growth and metastasis. Utilization of the “repertaxin” a small-molecule inhibitor of CXCR1 significantly reduces the breast CSC population, leading to decreased tumourigenicity and metastasis. MSCs are also known to influence breast cancer cells via the activation of the NF-kB pathway. A number of cytokines, including IL-6 and IL-8, secreted by MSCs are regulated by NF-kB and generate a positive feedback loop to maintain a chronic inflammatory state in tumour cells (Iliopoulos et al., 2010). Interestingly, this loop involves the mircro-RNA let7 as well as Lin28, a factor involved in breast CSC self-renewal and expansion. NF-kB activity in breast cancer metastasis is likely induced by infiltrating stromal cells (Tan et al., 2011). NF-kB has also been implicated in the regulation of mouse mammary stem cells during pregnancy. Elevated levels of progesterone during pregnancy induce RANK ligand (RANKL) in differentiated breast epithelial cells. RANKL in turn stimulates breast CSC self-renewal via activation of NF- kB in these cells (Asselin-Labat et al., 2010; Joshi et al., 2010). The increased incidence of aggressive breast cancers associated with pregnancy (Peck et al., 2002) may result from activation of similar pathways in breast CSCs (Asselin-Labat et al., 2010; Joshi et al., 2010). TGF-β potently induces expansion of breast CSCs by inducing transcriptional changes mediated by several key transcription factors. Several studies have demonstrated that paracrine TGF-β1 secreted by MSCs regulates the establishment of EMT in breast cancer cells by targeting the ZEB/miR-200 regulatory loop (Xu et al., 2012). A dramatic increase in the expression of EMT-specific genes in breast cancer cell lines has been observed following exposure to MSC-secreted factors, including TGF-β1 and TNF-α (Asiedu et al., 2011). These results suggest that MSCs may promote breast cancer metastasis by stimulating EMT-mediated expansion of CSCs, leading to tumour recurrence and drug resistance.
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