A prominent environmental stimulus of tumor dissemination is hypoxia, triggered by a high demand for cell proliferation and insufficient angiogenesis. Comparable to this process, hypertrophic AT expansion during obesity can also trigger local hypoxia (32
) that can further progress to AT fibrosis (33
). These obesity-related pathophysiological changes can lead to an environment that is conducive to cancer growth, such as chronic inflammation, inadequate angiogenesis, and enhanced fibrosis (8
). In this setting, obesity may contribute to an ETP-rich tumor microenvironment through a positive feed-forward mechanism. Indeed, COL6α3 message levels are upregulated in obese AT (34
). COL6 upregulation has been reported in various aspects of tumor progression. Malignant cancer cells can also express COL6; this has been reported for the mammary gland (18
), the colon (35
), pancreatic ductal adenocarcinomas (37
), and hepatocarcinomas (38
). Thus, the source of ETP in the tumor microenvironment may be heterogeneous, with signals cooperatively influencing cancer cell behavior through paracrine and autocrine pathways. Nevertheless, stromal adipocytes represent a prominent source for COL6 in the mammary tumor microenvironment (Supplemental Figure 10A). However, the detailed mechanisms underlying the specific effects of COL6 on tumor behavior have not fully been elucidated. Here, we have highlighted that ETP, a cleaved product of COL6, is likely to be a critical mediator of several tumor-associated phenomena and is of particular importance in tumor progression in the context of obesity.
Within the tumor milieu, EMT is initiated by extracellular stimuli. This can be exerted by ECM components (collagens, fibronectin, hyaluronic acids, and MMPs) as well as by certain growth factors (TGF-β, EGF, and HGF), all of which are provided by both paracrine and autocrine signals within the tumor microenvironment (25
). One of the prominent ECM molecules released from stromal adipocytes is COL6. As a COL6 processing product, ETP plays an important role in the local microenvironment, stimulating TGF-β–dependent EMT in the context of mammary tumors to potentiate prometastatic effects (Figure ). Gene expression profiling and immunostaining of tumor tissues from PyMT/ETP mice confirmed enhanced ETP-mediated acquisition of EMT characteristics, whereas in vitro data indicated that ETP alone did not induce EMT (data not shown). This suggests that ETP may function as an important costimulator of existing pathways for the EMT, such as TGF-β signaling and possibly activation of integrins and Wnt signals.
Increased tissue fibrosis, combined with high tissue rigidity (due to ECM remodeling and crosslinking), is positively associated with tumor growth (39
). Our results revealed an ETP-induced fibrotic environment, with high levels of myofibroblast accumulation within tumor tissues, as a key characteristic of ETP action. These activated myofibroblasts in ETP+
-tumors were derived, at least in part, by EMT. However, we cannot rule out that ETP may facilitate additional processes, such as microfibril assembly of preexisting collagen fibers (15
) or stimulation of myofibroblast differentiation (40
). Moreover, promoting transformed mesenchymal cell proliferation can enhance the appearance of additional stromal cells (41
); ETP may also effectively promote this process. Indeed, blocking the EMT by using a TGF-β neutralizing antibody did not completely eliminate fibrosis in ETP+
-tumors (Figure F). These data indicate that the ETP-induced EMT and subsequent fibrotic traits in tumors contribute to an increase in tumor growth and metastasis, which highlights a central role for ETP in tumor progression.
We have also provided evidence for the potent ETP-mediated chemoattractant properties. These ETP effects can even be mimicked in a tumor-free environment. A number of reports highlight significant correlations between COL6α3 and chronic inflammation, based on increased macrophage infiltration into AT depots of obese subjects (42
). We propose that the ETP-mediated chemoattractant properties described herein may offer a mechanistic basis for these clinical correlations. Neutralizing these ETP-mediated effects in normal, tumor-free AT may yield beneficial outcomes as well. Our current efforts are directed toward adipocyte-derived overexpression of ETP, which will answer the question of whether a local excess of ETP will exert beneficial effects (due to its proangiogenic properties) or negative effects (due to its proinflammatory and profibrotic properties) on a fat pad not challenged with an invading tumor.
We have limited insights into the stepwise process that leads to cleavage of ETP from its parent molecule. We do not know which proteases are involved, or from where they originate. Fibrosis in obese AT is associated with an increase of various MMPs or TIMPs resulting in collagen degradation (44
). MMP-11, MMP-2, and MMP-9 have been suggested as peptidase for COL6 (45
), although there is no further evidence whether these MMPs cleave ETP. Base on the fact that most cancer cells express high levels of MMPs associated with tumor growth and metastasis (47
), it is likely that there are abundant sources for ETP cleavage activity within the tumor microenvironment. The identification of the critical protease involved in ETP processing may offer a new approach to curbing growth by pharmacologically inhibiting this step. Our findings unveiled an important role of the adipocyte as an active component of the tumor stroma that actively interacts with cancer cells and a number of other relevant local cell types. Our data highlight that an adipocyte-derived ECM cleavage product actively contributed toward the remodeling of the tumor microenvironment by enabling the progression of tumor growth and metastasis through enhancement of the EMT process and subsequent chemotaxis of endothelial cells and macrophages. In many aspects, the deposition of ECM components, such as ETP in the tumor stroma, resembles a wound-healing process, as this involves the recruitment and stimulation of immune cells, endothelial cells, and fibroblasts during the wound repair process. However, unlike during the wound-healing process, ETP prompts cancer cells to sustain mesenchymal cell–like traits and activates fibroblasts in the tumor stroma, drastically increasing local fibrosis and eventually enhancing metastatic growth. Our findings have further implications for several tissues that have an associated pathological fibrotic component, such as the liver (48
), cartilage (49
), lung (50
), and heart (51
); COL6 expression has been documented in all these tissues. Future efforts targeted toward ETP neutralization in various pathological settings should establish whether this approach is a viable antifibrotic strategy that is generally beneficial, not only in the setting of tumor progression and metastasis, but also during normal AT expansion.