Breast cancer is the second leading cause of cancer death in women. Although the 5-year survival rate for women with localized disease is high at 98%, this number drops precipitously to only 27% once the primary tumor has metastasized [45
]. For this reason, it is vitally important for science and medicine to enhance their understanding of the processes that underlie breast cancer invasion and metastasis. It has long been established that breast cancer development reflects a loss-of-tissue organization and differentiation, factors that have more recently been associated with increases in LOX expression and activity [46,47
]. We show here that TGF-β and EMT both induce the expression and secretion of LOX from normal and malignant MECs and that mammary tumors engineered to house elevated TGF-β signaling produced more LOX than did their parental counterparts, which correlated with the ability of TGF-β to stimulate mammary tumor growth and pulmonary metastasis [8
]. Equally important, we provide the first evidence that the manner in which MECs respond to TGF-β can be regulated by tissue rigidity, which elicits dramatic changes in MEC acinar morphology and growth in a LOX-and hydrogen peroxide-dependent manner. Although the exact mechanisms whereby tissue tension regulates TGF-β1 function remain to be elucidated fully, it is tempting to speculate that enhanced ECM rigidity may promote the inappropriate clustering of TGF-β receptors with integrins and other growth factor receptors, thereby inducing amplified coupling of TGF-β to its noncanonical effectors [48,49
]. Accordingly, we demonstrated that antagonizing LOX activity led to the diminished ability of TGF-β to stimulate MEC invasion and EMT. Furthermore, we show that antagonizing LOX activity partially uncoupled TGF-β1 from p38 MAPK activation in metastatic 4T1 cells, whereas only catalase administration facilitated this event in normal NMuMG cells, suggesting that the roles of LOX and hydrogen peroxide depend on the pathophysiology of MECs.
It is important to note that although our current findings support an extracellular role of LOX in mediating the oncogenic activities of TGF-β (i.e., catalase neutralizes hydrogen peroxide), we cannot exclude the possibility that LOX may promote oncogenic TGF-β signaling by acting intracellularly. Indeed, mature LOX has been detected not only in the ECM but also in the cytoplasm () and nucleus of malignant cells [10,11
]; however, the identification of specific molecules capable of interacting with and/or being targeted by LOX in either intracellular compartment remains to be elucidated fully [11
]. Along these lines, LOX was shown to activate Src and promote cell adhesion through a hydrogen peroxide-dependent mechanism [23
]. We show here that administering catalase to degrade hydrogen peroxide prevented TGF-β from stimulating MEC proliferation, EMT, and invasion () and from fully activating p38 MAPK (). Thus, hydrogen peroxide may function as a novel “second messenger” for TGF-β in normal and malignant MECs. Collectively, our findings suggest that LOX may play an important role in initiating the conversion of TGF-β function from a suppressor to a promoter of mammary tumorigenesis. At present, the specific players targeted by LOX and hydrogen peroxide that affect TGF-β signaling remain an active and important topic for future experimentation. In addition, it is unclear what overlapping functions other LOX family members may play. Importantly, LOXL2 is not inhibited by βAPN treatment and may compensate for loss of LOX function [11,50
], and in fact, both LOX and LOXL2 mediate the ability of HIF-1α to suppress E-cadherin expression [51
]. Thus, future studies need to dissect the relative contribution of individual LOX family members to the initiation of oncogenic TGF-β signaling and its coupling to canonical and non-canonical effectors.
LOX plays a critical role during the formation of premetastatic niches by stimulating collagen cross-linking and fibronectin synthesis, leading to the recruitment of bone marrow-derived cells to metastatic niches [13
]. TGF-β has also been implicated in the recruitment of immature bone marrow-derived cells to drive breast cancer metastasis [52
], which suggests a potential link between TGF-β and LOX in regulating the formation of premetastatic niches. Interestingly, the use of copper chelators in preclinical and phase 2 clinical trials has shown some success in diminishing metastatic burden [53
], findings that are potentially important because LOX activity is absolutely dependent on copper as one of its two cofactors (the other being lysyl tyrosyl quinone [11
]). Thus, it is plausible that the clinical success of copper chelators to reduce tumor metastasis lies in their ability to inhibit LOX activity and, consequently, perhaps to alleviate the oncogenic activities of TGF-β as well. Along these lines, tumor hypoxia predicts for poor prognosis and decreased survival of breast cancer patients, which is linked to hypoxia-induced expression of LOX and the generation of metastatic niches in breast cancer patients [13,16,54
]. These findings, together with those presented herein, support the idea that LOX dictates how malignant MECs respond to the varied activities of TGF-β, and as such, identify LOX as a novel participant in oncogenic TGF-β1 signaling in late-stage mammary tumors. Thus, chemotherapeutic targeting of LOX may offer new inroads to alleviate breast cancer progression stimulated by TGF-β.
Lastly, the ability of LOX to cross-link collagen to elastin results in increased tissue tension and ECM rigidity [12,15,39,55
]. More recently, ECM rigidity has been shown to play an important role in breast cancer development, particularly their acquisition of invasive and metastatic phenotypes [12,39,47,56
]. The ability of normal and malignant MECs to sense ECM stiffness transpires through integrins and other mechanotransducers, which in turn activate Src, FAK, and the GTPases, Rho, Rac, and Cdc42 [46,47
]. Importantly, we identified an oncogenic TGF-β signaling axis comprised in part of αv
integrin, FAK, and Src that induces mammary tumor growth, invasion, and metastasis in mice [4,8,9
], as well as stimulates significant LOX expression in these same mammary tumors (). We speculate that tumor-initiated MECs evolve in compliant microenvironments that favor canonical Smad2/3 signaling stimulated by TGF-β. The continued growth of the developing neoplasm enhances ECM rigidity by upregulating TGF-β production () and LOX expression (), which may lead to the inappropriate formation of integrin-TβR-II complexes [6–8
]. Once formed, these complexes are also likely to interact with other growth factor receptors that presumably amplify the activation of noncanonical effectors by TGF-β [57
]. Ultimately, these adverse events culminate in the ability of TGF-β to induce the acquisition of EMT, stem-like, and metastatic phenotypes in malignant MECs, leading to their metastasis at distant locales. Moreover, extending our findings to encompass sites of micrometastases [13
], which are predicted to possess compliant ECM tension, leads us to propose that the cytostatic activities of TGF-β may be partially reinstated at these newly seeded sites, perhaps contributing to tumor dormancy. Over time, this vicious micro-environmental cycle is repeated, leading to disease recurrence and poor clinical outcomes in breast cancer patients harboring metastatic disease. The basic tenets of this model are supported by the findings presented herein (), and as such, this model should serve as a launching point for future studies aimed at identifying the individual effectors operant in regulating ECM tension and TGF-β1 function in distinct breast cancer subtypes.