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Pathological neovascularization relies on an imbalance between potent proangiogenic agents and equally effective antiangiogenic cues. Collectively, these factors contribute to an angiogenic niche within the tumor microenvironment. Oncogenic events and hypoxia contribute to augmented levels of angiokines, and thereby activate the so-called angiogenic switch to promote aggressive tumorigenic and metastatic growth. Soluble decorin functions as a paracrine pan-inhibitor of receptor tyrosine kinases, such as Met and epidermal growth factor receptor, and thus is capable of suppressing angiogenesis under normoxia. This leads to noncanonical repression of hypoxia-inducible factor 1-alpha and vascular endothelial growth factor A (VEGFA), and concurrent induction of thrombospondin-1. The substantial induction of endogenous tumor cell-derived thrombospondin-1, a potent antiangiogenic effector, led us to the discovery of an unexpected secretory phenotype occurring very rapidly (within 5 min) after decorin treatment of the triple-negative basal breast carcinoma cell line MDA-MB-231. Surprisingly, the effect was not mediated by Met receptor antagonism, as initially hypothesized, but required epidermal growth factor receptor signaling to achieve swift and robust thrombospondin-1 release. Furthermore, this effect was ultimately dependent on the prompt degradation of Ras homolog gene family member A, via the 26S proteasome, leading to direct inactivation of Rho-associated coiled-coil containing protein kinase 1. The latter led to derepression of thrombospondin-1 secretion. Collectively, these data provide a novel mechanistic role for Rho-associated coiled-coil containing protein kinase 1, in addition to providing the first conclusive evidence of decorin exclusively targeting a receptor tyrosine kinase to achieve a specific effect. The overall effects of soluble decorin on the tumor microenvironment would cause an immediately-early as well as a sustained antiangiogenic response in vivo.
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Rho-associated coiled-coil containing protein kinase 1
receptor tyrosine kinase
standard error of the mean
small interfering RNA
small leucine-rich proteoglycan
vascular endothelial growth factor A
An intricate balance exists between proangiogenic and antiangiogenic factors, which either promote or impede, respectively, pathological angiogenesis within the tumor microenvironment . When this balance is altered, as a consequence of oncogenesis, to be in favor of proangiokines, an angiocompetent milieu is formed via the so-called angiogenic switch [2-6]. This pathobiological imbalance within the tumor microenvironment allows for neovascularization of the tumorigenic tissue, leading to enhanced aggressive behavior, increased metastasis, and poorer prognosis . This balance of angiocrine factors is intimately orchestrated by the plethora of functions exerted by the surrounding extracellular matrix constituents that are able to differentially regulate angiogenesis, and often are the critical determinants of this .
Proteoglycan-mediated control of tumor angiogenesis exemplifies this principle [8-10]. Of particular interest are the inherently powerful antiangiogenic properties exerted by the small leucine-rich proteoglycan (SLRP) decorin [11, 12], a paradigmatic member of the SLRP superfamily. Decorin, originally identified as a regulator of type I collagen fibrillogenesis and [13-20] and as the product of a highly induced gene in the stroma of human colon carcinomas [21, 22], is encoded by a large and highly conserved gene with a complex promoter structure [23-25]. Soluble, monomeric decorin  certainly functions in a paracrine manner to bind, with high affinity, multiple receptor tyrosine kinases (RTKs), including members of the ErbB family, such as the epidermal growth factor receptor (EGFR) and ErbB4 [27-29], and has been identified as the only antagonistic ligand of Met, the hepatocyte growth factor (HGF) receptor [30, 31]. In addition, decorin binds to the insulin-like growth factor receptor I [32-34] and attenuates its activity in bladder cancer cells . These diverse bioactivities mean that decorin has highly effective antitumorigenic [31, 36-39] and antimetastatic [40, 41] properties. Decorin is also involved in innate immunity and hypersensitivity reactions [42-44], and in bone pathophysiology . Genetic ablation of the decorin gene favors lymphomagenesis in a p53-null background , induces intestinal tumor formation in a C57BL/6 background [47, 48]. Decorin also plays a role in wound healing . Concurrent with these activities is the induction of cyclin-dependent kinase inhibitors such as p21WAF1 [50, 51], together with the degradation, in a noncanonical fashion, of potent oncoproteins such as β-catenin and Myc . Therefore, decorin acts as a paracrine tumor repressor by acting as a pan-RTK inhibitor at the cell surface of tumor cells [10, 53].
Decorin represses proangiogenic factors (hypoxia-inducible factor 1-alpha and VEGFA and simultaneously causes transcriptional induction of antiangiogenic molecules such as tissue inhibitor of metalloprotease 3 and thrombospondin (TSP)-1 under normoxia via suppression of proangiogenic HGF–Met signaling [11, 54-56]. Previous work  demonstrated an acute transcriptional response for THBS1 expression that correlated with increased TSP-1 levels in the triple-negative basal breast carcinoma cell line MDA-MB-231, as well as in tumor xenograft models composed of the same cell type. TSP-1 is an archetypical matricellular component, encoded by a member of a gene family that encodes five large, modular, calcium-binding, secreted glycoproteins . TSP-1 is a long, filamentous protein that is capable of binding several cell surface receptors, enabling diverse regulation of cellular function among many different cell types . Although it was originally identified as a secreted monomeric glycoprotein of ~ 140 kDa, TSP-1 functions primarily as a trimer, and is derived from thrombin-stimulated platelets and platelet α-granules, accounting for ~ 3% and ~ 25% of total protein content, respectively . It is now well established that TSP-1 is expressed by a wide variety of cell types, but it is predominantly expressed by vascular smooth muscle cells and endothelial cells . Functionally, TSP-1 inhibits wound healing, inhibits matrix metalloproteinase-9 and VEGFA liberation, triggers endothelial cell apoptosis via engagement of CD36 and signaling via Jun N-terminal kinase and p38 stress-activated protein kinases, and modulates adhesion [58-60]. Additional functions of TSP-1 include regulation of NO–cGMP signaling via engagement and ligation of CD47 with vascular endothelial growth factor receptor-2 within the cardiovascular system , regulation of synaptogenesis in the central nervous system , and modulation of transforming growth factor-β activation and fibrosis  and wound healing . Moreover, TSP-1 inhibits angiogenesis via a direct effect on endothelial cell migration and survival, and by affecting VEGFA availability and vascular endothelial growth factor receptor-2 activity [65, 66]. Notably, TSP-1-deficient mice show a lordotic curvature of the spine, increases in the number of circulating monocytes and eosinophils, and pulmonary inflammation . Interestingly, the TSP-1 null mouse was not embryonic lethal, perhaps because of redundancy among the other TSP gene members .
In the context of cancer, oncogenic Ras signaling  and altered Myc activity, downstream of Ras , combinatorially repress TSP-1 expression. The transcriptional inhibitor Id1 was recently shown to repress THBS1 expression, as Id1 deficiency is associated with increased TSP-1 levels . As decorin is capable of unconventionally downregulating Myc , and there is a concept that Myc drives Id1 induction , we sought to further characterize the mechanism for decorin-mediated induction of TSP-1 in the MDA-MB-231 cell line, presumably downstream of Met . Unexpectedly, we found a prompt and robust secretory phenotype mediated by decorin that requires EGFR signaling, independently of Met, and that is orchestrated through the concerted degradation of Ras homolog gene family member A (RhoA) and subsequent inactivation of Rho-associated coiled-coil containing protein kinase 1 (ROCK1) to allow for rapid secretion of TSP-1 from MDA-MB-231 cells. Collectively, our results suggest a novel secretion-inducing role for decorin, and offer new perspectives regarding the ability of this SLRP to attenuate the proangiogenic niche of the surrounding tumor microenvironment.
Decorin induces rapid and biphasic release of TSP-1 in MDA-MB-231 breast carcinoma cells
We have previously described a function for exogenous soluble decorin in inducing the expression of THBS1 mRNA and protein in MDA-MB-231 cells following engagement of Met as part of a potent angiostatic program . Therefore, in the current study, we aimed to further characterize the mechanism of TSP-1 regulation and subsequent secretion under the influence of decorin. Analysis of tumor-conditioned medium (TCM) revealed rapid mobilization of TSP-1 in as little as 5 min (~ 1.7-fold) following decorin exposure (Fig. 1A,B). It should be noted that the decorin protein core was utilized throughout the study (hereafter referred to as decorin), unless otherwise indicated. Interestingly, the level of TSP-1 spiked at 10 min (~ 2.2-fold), and then returned to near baseline levels at 20 min post-treatment (Fig. 1B). However, the level of TSP-1 began to increase a second time at 30 min, and reached a plateau after 1 h (Fig. 1A,B). We believe that this biphasic release of TSP-1 reflects an initial burst of TSP-1 release, most likely from preformed TSP-1 stored in the cytoplasm, followed by enhanced expression of THBS1 mRNA for the second phase, in a manner analogous to platelet-derived growth factor-mediated induction of THBS1 .
To further corroborate these finding, we performed confocal microscopy, wherein MDA-MB-231 cells were exposed to decorin for 40 min and probed for TSP-1. The distribution of TSP-1 in control cells (Fig. 1C) showed primarily a uniformly granular pattern. TSP-1 staining, with regard to both the number of granules and the signal intensity of TSP-1 deposits in the decorin-treated cells, after 40 min was significantly diminished (Fig. 1D), in accordance with the above data showing rapid release of TSP-1 from the cell.
In contrast to the rapid release of secreted TSP-1 induced by decorin, there were no substantial changes in the intracellular stores of TSP-1 at early time points (5–20 min) (Fig. 1E,F). However, at 30 min, there was significant accumulation (~ 1.8-fold) of intracellular TSP-1. These intracellular levels of TSP-1 remained high at 1 and 2 h post-treatment (not shown).
Collectively, our results show an unexpected and novel role for decorin in inducing rapid, biphasic and time-dependent release of TSP-1 from quiescent (serum-starved) MDA-MB-231 cells.
Decorin requires EGFR signaling, but not Met, to induce rapid TSP-1 secretion
Next, we sought to identify the mechanism of this biological activity; that is, we tested which of the known decorin RTKs was responsible for coordinating this swift release of TSP-1 in response to soluble decorin. Our main hypothesis was that Met would be the primary receptor responsible for this activity, as Met has been previously implicated in coordinating the other aspects of decorin-mediated angiostasis . Therefore, we performed similar experiments in the presence or absence of the Met tyrosine kinase inhibitor SU11274, a highly selective inhibitor of the ATP-binding domain for various Met mutants . To this end, we pretreated MDA-MB-231 cells for 30 min with SU11274, and followed this with incubation with decorin for 10 min. Consistently, decorin facilitated a rapid burst of TSP-1 secretion into the medium; surprisingly, this effect was not blocked by SU11274 (Fig. 2A), as decorin still induced TSP-1 secretion (approximately twofold). Quantification of TSP-1 secretion in SU11274-pretreated cells as well as in cells treated with decorin alone revealed marked increases in TSP-1 levels in both cases (P < 0.001; Fig. 2C), but no significant differences between the two experimental conditions (P = 0.21). Next, in an attempt to link these effects to Met, we pretreated MDA-MB-231 cells with HGF for 30 min . Decorin was able to induce secretion, whereas HGF pretreatment failed to block decorin-mediated secretion of TSP-1 (Fig. 2B). Quantification of secreted TSP-1 revealed significant induction of TSP-1 secretion in the presence or absence of HGF (P < 0.001; Fig. 2D) and no significant differences between the two experimental conditions (P = 0.35). Collectively, these results indicate that decorin engagement of Met is not involved in the secretion of TSP-1 by MDA-MB-231 cells.
The original identification of decorin-mediated antagonism towards RTKs began with EGFR; upon decorin binding, there is receptor dimerization, and rapid trans-autophosphorylation reminiscent of the effect on Met within 10 min . We therefore investigated the role of EGFR signaling in TSP-1 secretion, as MDA-MB-231 cells express EGFR, but not HER2/Neu. When immunofluorescence was used to probe TSP-1 secretion in response to decorin, pretreatment with the EGFR small tyrosine kinase inhibitor AG1478  abrogated the ability of decorin to deplete TSP-1 (compare Fig. 2F with Fig. 2G), making control (Fig. 2E) and AG1478-pretreated cells look almost indistinguishable. Furthermore, application of the EGFR-specific monoclonal blocking antibody mAb425  blocked decorin-mediated secretion of TSP-1 and mimicked control conditions as determined by immunofluorescence. Application of either AG1478 or mAb425 prior to decorin treatment almost completely restored the punctate pattern of TSP-1 to control signatures, and significantly obviated the ability of decorin to induce TSP-1 release, as determined by the average TSP-1 fluorescence signal (Fig. 2I).
Taken together, these data show the ability of decorin to differentially signal and integrate specific aspects of the angiostatic program across different RTKs expressed by the tumor cell. Thus, rapid secretion of TSP-1 is almost exclusively dependent on EGFR, but not Met, in these basal breast carcinoma cells.
Rapid secretion of TSP-1 depends on ROCK1 inhibition
The role of p160-ROCK1 in the control of TSP-1 has been evaluated, and it has been assigned an inhibitory role as a downstream effector of the Ras–phosphoinositide 3-kinase–RhoA signaling pathway to achieve THBS1 transcriptional repression . Indeed, treatment with the ROCK1 inhibitor Y27632 derepresses TSP-1 repression following an 8-h treatment . Suppression of ROCK1 by a variety of mechanisms reduces cancer metastasis and invasion in lung adenocarcinoma  and gastric cancer via miRNA-148a . However, investigation of the role of ROCK1 in regulating secretion has been limited and, surprisingly, has focused on the effects of ROCK1 inhibition functionally linked to hyperinsulinemia . Therefore, we hypothesized that Y27632, a specific small molecule inhibitor of ROCK1, would be able to recapitulate the pattern of rapid decorin-induced TSP-1 release from MDA-MB-231 cells. Incubation of MDA-MB-231 cells with Y27632 alone induced rapid and robust secretion of TSP-1, starting as early as 5 min, and peaking at 10 min (~ 2.7-fold), with a plateau (~ 2.5-fold) that was sustainable for up to 2 h after treatment with Y27632 (Fig. 3A,B). These results recapitulate the decorin-induced secretion of TSP-1 with comparable kinetics (Fig. 1A,B). Interestingly, no significant modulation of ROCK1 protein levels was seen with decorin treatment for up to 30 min (Fig. 3C,D) or up to 2 h (data not shown). Potentially via RhoA, the activity of ROCK1 must be suppressed to allow for rapid TSP-1 secretion as this is independent of ROCK1 protein levels.
Next, we established that ROCK1 inhibition was the primary effector for decorin-mediated TSP-1 release, as treatment with either decorin or Y27632 alone led to maximal TSP-1 secretion after 10 min (Fig. 3E,F). Furthermore, pretreatment with Y27632 followed by decorin application did not induce further liberation of TSP-1, indicating that ROCK1 inhibition is the primary target for decorin to induce rapid TSP-1 mobilization (Fig. 3E,F).
The role of ROCK1 was functionally tied to the control of TSP-1 secretion through small interfering RNA (siRNA)-mediated knockdown of ROCK1 (denoted as siROCK1). We achieved significant depletion of ROCK1 as detected by immunoblotting (> 85% of control levels; Fig. 3G) as compared with siScramble (siScram)-transfected controls. The amount of secreted TSP-1 in the presence of siROCK1 alone was significantly increased relative to siScram controls, indicating a role for ROCK1 in suppressing basal TSP-1 secretion (Fig. 3H,I), and consistent with the above results showing that inhibition with Y27632 increased the secretion of TSP-1. Finally, decorin was unable to induce a further increase in the amount of secreted TSP-1 in the presence of siROCK1 (Fig. 3H,I). These results indicate and further confirm that ROCK1 is epistatic to the bioactivity of decorin-mediated secretion of TSP-1, and that, upon inhibition or depletion of ROCK1, secreted TSP-1 is rapidly released.
Collectively, these data show a novel role for ROCK1 in controlling the basal levels of a highly antiangiogenic molecule. Furthermore, ROCK1 serves as the main downstream effector in the mechanism of decorin-induced TSP-1 release that results from positive EGFR signaling.
Decorin depends on EGFR to rapidly degrade RhoA and to induce TSP-1 secretion
The cellular role of ROCK1 is activated and thus orchestrated by direct and putative binding of RhoA , a small GTPase implicated in motility, invasion, and proliferation , as well as in promoting oncogenic transformation and a more aggressive tumor phenotype . Furthermore, it has been found that increased RhoA and Ras homolog gene family member C expression ultimately correlates with poorer prognoses in breast cancers . Thus, as inhibition of ROCK1 with Y27632 even in the presence of decorin did not synergistically or additively increase the amount of total secreted TSP-1, and there was a lack of ROCK1 modulation at the protein level as assayed for up to 2 h, we hypothesized that ROCK1 would be unable to be activated via antagonism of RhoA, upon decorin engaging EGFR. Immunoblotting revealed a stark reduction (< 50%) in the amount of RhoA in MDA-MB-231 cells within 10 min of decorin treatment (Fig. 4A). This observation supports the hypothesis that ROCK1 fails to become activated because of a pronounced reduction in RhoA levels. This effect was sensitive to EGFR kinase inhibition, as pretreatment with AG1478 followed by decorin (10 min) failed to reduce total RhoA levels as compared with decorin-treated samples (Fig. 4B). This effect was further verified following quantification, which showed a decorin-dependent decrease (Fig. 4B) in the RhoA level (by > 60%) that was wholly abrogated by pretreatment with AG1478 (Fig. 4B). Moreover, pretreatment with the EGFR monoclonal blocking antibody mAb425 elicited identical effects by abolishing suppression of RhoA as compared with decorin alone (Fig. 4C). This rapid decrease (within 10 min of decorin application) in the RhoA level is the result of active proteolysis via the 26S proteasome, as lactacystin, a powerful proteasome inhibitor, was able to completely abolish the decorin-induced degradation of RhoA (Fig. 4D).
Taken together, these data show a prompt and vigorous decorin-mediated response to degrade RhoA via the 26S proteasome. RhoA degradation is dependent on both EGFR signaling and the caveat that decorin binds EGFR, and not Met.
Decorin depends on classic secretory pathways for rapid TSP-1 secretion
Exosomes constitute an emerging class of secreted, membrane-bound vesicles that allow for intercellular communication by delivering a host of bioactive molecules, including proteins, lipids, RNA, and DNA, that seem to have an immunosuppressive role . These nanovesicular bodies have been shown to play functionally important roles in both the initiation and progression of pathobiological states, including cancer. Interestingly, tumorigenic cells secrete increased amounts of exosomes relative to their nontumorigenic counterparts, and show a distinct proteomic signature that is representative of the host cell . We therefore hypothesized that exosomes might be responsible for the rapid en masse secretion of TSP-1 from MDA-MB-231 cells. To investigate this, we subjected tumor cell-conditioned medium, following incubation with decorin for 6 h, to differential ultracentrifugation to isolate exosomal fractions, and followed this with immunoblotting for the detection of exosomal TSP-1. It is important to note that the decorin treatment time was significantly increased to allow for maximal collection of exosomes from the medium. Thus, with caveolin-1 (Cav-1) as an exosomal marker , we found no significant change between control and decorin-treated exosomal isolates (Fig. 5A). Quantification of the results of three independent experiments normalized to the fetal bovine serum detected in the medium showed no significant changes in TSP-1 levels (Fig. 5B). Paradoxically, exosomal Cav-1 was increased relative to control (Fig. 5A), suggesting that decorin triggers an increase in exosomal secretion, but that this does not underlie the secretory mechanism for TSP-1.
Classic secretion from the cell depends on the movement of vesicles via the microtubular network, followed by fusion with the plasma membrane and secretion of the contents to the extracellular space. Thus, preincubation of MDA-MB-231 cells with nocodazole, which interferes with microtubule polymerization, was able to block decorin-mediated release of TSP-1 (Fig. 5F) as compared with decorin alone (Fig. 5D), and the amount of TSP-1 release was similar to that in the control (Fig. 5C). There was a small, but significant, reduction in TSP-1 levels following pretreatment with nocodazole and subsequent decorin incubation (P = 0.041; Fig. 5G), as compared with controls, suggesting that there are alternative release mechanisms. However, as compared with decorin alone, a significant block (P = 0.009; Fig. 5G) occurred, indicating a requirement for microtubules for mediating the release of TSP-1 under the influence of decorin. Finally, RhoA is potently degraded by decorin via the 26S proteasome (Fig. 4D), and this has functional consequences for the ability of decorin to trigger release of TSP-1. As shown in Fig. 5E, pretreatment with lactacystin (which attenuates RhoA degradation) was able to hinder the decorin-mediated release of TSP-1, suggesting a requirement for RhoA degradation as part of the mechanism to allow for accelerated TSP-1 release. Indeed, quantification of TSP-1 fluorescence revealed an almost complete block of TSP-1 release as compared with decorin (P = 0.001; Fig. 5G), the amount of TSP-1 release being almost identical to that in the control (P = 0.29; Fig. 5G).
Overall, we have demonstrated that decorin utilizes the classic secretory pathway, which is dependent on microtubules, to elicit TSP-1 secretion. Furthermore, this pathway is profoundly sensitive to the levels of RhoA, as prevention of degradation of this cytoskeletal mediator completely blocked decorin-triggered release of TSP-1.
A fragile balance among the angiokine signaling factors, as well as the tumor parenchyma and endothelial cells residing within the tumor microenvironment, mediates the tumor angiogenic switch. Angiogenesis critically relies on matrix-derived cues for coordination, particularly SLRPs . From the perspective of the tumor proper, decorin is a potent inhibitor of tumor angiogenesis . Recently, we found potent transcriptional repression of numerous genes encoding soluble proangiogenic mediators responsible for the initial sculpting and continued maintenance of the angiogenic response, such as VEGFA, FGF2, and ENG, with concurrent induction of genes encoding soluble antiangiogenic effectors, including THBS1 and TIMP3 . These data demonstrate, for the first time, potent transcriptional induction of soluble angiostatic gene products from MDA-MB-231 cells in the presence of decorin in both in vitro and in vivo models of triple-negative breast carcinoma .
In the present study, we have extended our investigations on the underlying mechanism of decorin-mediated angiostasis by further characterizing TSP-1 regulation, as this matricellular component has a profound influence on the state of the angiogenic niche by compromising the viability of endothelial cells . We discovered a novel mechanism for the control of TSP-1 secretion (Fig. 6). In contrast to the protracted transcriptional response of THBS1 and TSP-1 that occurs following an 8-h incubation with decorin , we found very rapid secretion (within 5 min) of presumably preformed TSP-1 from MDA-MB-231 cells that appears to be biphasic in nature. The second phase of TSP-1 release might be mediated via the induction of THBS1 mRNA expression or the consequent accumulation of the TSP-1 oligomeric form within the endoplasmic reticulum. As cellular TSP-1 begins to increase, this is consistent with the ~ 30-min maturation time for TSP-1 accumulation within this compartment. These data show an immediate transition from the tumor parenchyma to start reprogramming the otherwise proangiogenic niche.
We initially hypothesized that this effect would be coordinated by Met, as decorin, which behaves as a partial agonist of Met, triggers rapid phosphorylation of the Met catalytic domain (at Tyr1234/5) within 5–10 min before phosphorylation returns to baseline . Rapid TSP-1 release might be related to this spike in phosphorylation of the intracellular domains of Met immediately prior to receptor internalization. However, this was found to not be the case. Instead, decorin requires signaling via EGFR, as AG1478 and the function-blocking antibody mAb425 abrogated the immediate-early release of TSP-1 (Fig. 6). This is consistent with the role of decorin in triggering EGFR phosphorylation upon binding. This phosphorylation signature allows for the swift release of TSP-1 from MDA-MB-231 cells. This effect is intriguing, insofar as this particular cell type expresses both Met and EGFR; however, decorin coordinates specific cellular events, depending on the receptor that it engages, and thus integrates the signal over the repertoire of receptors known to bind decorin. This is thought to be mediated by the differential binding affinities that decorin has for EGFR and Met (Kd values of ~ 80 and 2 nm, respectively). Despite the fact that decorin has a higher binding affinity for Met than that of EGFR, we have clearly shown that Met does not play a role in TSP-1 release. Intrinsic differences between EGFR and Met may influence decorin binding to EGFR, such as the topological requirements conferred by EGFR and/or the manifestation of fundamentally different phosphorylation patterns from that of Met to allow for TSP-1 release. Domain-swapping experiments to precisely identify the bioactive determinants responsible for these differences between EGFR and Met will be of great importance for understanding this phenomenon. Furthermore, these data establish, for the first time, a role of decorin to not only bind, but also required to signal via an RTK to elicit downstream cellular events to achieve angiostasis.
Our results indicate that decorin-induced TSP-1 release is not attributable to enhanced early transcriptional activity, as TSP-1 levels peaked within 10 min of stimulation. Thus, we began to analyze downstream effectors of EGFR that could coordinate TSP-1 release. It has been established that Ras–RhoA–ROCK1 signaling is important in the control of Myc to repress THBS1 . Increases in general protein secretion have been achieved through the inhibition of ROCK1 , and this has also been found to be the case for decorin-induced TSP-1 secretion, as no further secretion of TSP-1 was found to occur either upon small molecule inhibition or siRNA-mediated silencing of ROCK1. Furthermore, as no apparent changes in total ROCK1 levels occurred, we found prominent degradation via the 26S proteasome of RhoA, the key small GTPase that activates ROCK1, to be required for this bioactivity (Fig. 6). Moreover, blocking the degradation of RhoA prevented decorin-dependent release of TSP-1. Consistent with the finding of EGFR-dependent signaling, blocking with AG1478 also prevented RhoA degradation. These data identify a further antimetastatic role for decorin, not only through powerful suppression and degradation of RhoA, but also through derepression of potent angiostatic agent secretion in a noncanonical RhoA/ROCK1-dependent manner. Therefore, immediate-early loss of RhoA would presumably preclude activation of ROCK1, leading to the swift derepression of TSP-1 release, and thus provide a mechanistic basis for this phenomenon downstream of EGFR signaling.
Finally, we have demonstrated that TSP-1 release does not involve secretion of TSP-1 from nanovesicles such as exosomes, but does depend on a stable microtubule network, which is very sensitive to the levels of RhoA and is dependent on EGFR. This has several implications for the overall ability of decorin to impede tumorigenesis and metastatic capacity. As decorin requires competent microtubule polymerization to drive secretion of TSP-1 (Fig. 6), stable microtubule growth also has a very detrimental effect on cell motility and migration, as a decrease in microtubule stabilization leads to increased tumor cell invasion . Interestingly, this is dependent on the activity of p27Kip1 , a cyclin-dependent kinase inhibitor that is inducible by decorin . Therefore, rapid secretion of TSP-1 via RhoA/ROCK1 inhibition might be dependent on the activity of p27Kip1, thus leading to microtubule stabilization.
These data indicate an exceptionally early ability of decorin to begin to transform the surrounding tumor microenvironment by inducing secretion of soluble angiokine factors that inhibit tumor angiogenesis from the perspective of the tumor proper. Indeed, the profound effect that decorin has on the tumor microenvironment was further exemplified in a recent study that used systemic administration of decorin protein core to a triple-negative orthotopic breast carcinoma xenograft model . Decorin protein core was able to exclusively modulate stroma-specific (Mus musculus) genes without significant modulation of tumor xenograft (Homo sapiens) transcripts, resulting in a defined tumor microenvironment gene signature . It remains unclear how soluble decorin, which is canonically viewed as a pan-RTK inhibitor acting on the tumor proper, is able to reprogram the surrounding tumor microenvironment through transcriptional induction of potent tumor suppressor genes. It is conceivable that decorin, acting on the tumor proper via RTK engagement, is able to prime the tumor microenvironment through the release of antiangiogenic mediators to initiate this signature obtained from transcriptomic profiling. This might be of the utmost importance for the contribution of endothelial cell-mediated angiogenesis following TSP-1 binding to CD36 and CD47.
In conclusion, our data further establish decorin as a key inhibitor of tumorigenesis by contributing to the so-called ‘tumor secretome’, and reducing the ability of the tumor proper to initiate and sustain competent angiogenic processes from the stroma.
Cells, inhibitors, and antibodies
MDA-MB-231 triple-negative breast carcinoma cells were obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in DMEM supplemented with 5% fetal bovine serum (SAFC Biosciences, Lenexa, KS, USA) and 100 μg·mL−1 penicillin/streptomycin (MediaTech, Manassas, VA, USA). Primary antibodies against ROCK1 (H-85), used at a 1 : 1000 dilution, RhoA (26C4), used at a 1 : 500 dilution, and Cav-1 (1-C), used at a 1 : 500 dilution, were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); primary mouse monoclonal antibody against TSP-1 (1 : 500) was from Abcam (Boston, MA, USA). Secondary antibodies [horseradish peroxidase (HRP)-comugated goat anti-(rabbit IgG) (AP307P) and HRP-conjugated donkey anti-(mouse IgG) (AP192P)] were from Millipore (Billerica, MA, USA), and were used at 1 : 2000 and 1 : 6000 dilutions, respectively. HGF (H9661), lactacystin (L6785) and nocodazole (M1404) were from Sigma-Aldrich (St Louis, MO, USA). The Met kinase inhibitor SU11274 (448101), the ROCK1 inhibitor Y27632 (68800) and the EGFR kinase inhibitor AG1478 (658552) were from Calbiochem (Darmstadt, Germany). The mouse monoclonal EGFR-blocking antibody was a generous gift from U. Rodeck (Kimmel Cancer Center, Thomas Jefferson University). The purification of decorin protein core has been described extensively elsewhere [41, 91]. In brief, recombinant human decorin, as a poly-His6 fusion protein, was expressed in 293-EBNA cells. The 293-EBNA cells were serum-starved for maximal secretion of both decorin species: the glycanated and unglycanated forms of human decorin. This material was then passed through an Ni2+–nitrilotriacetic acid chelating column, and this was followed by exposure to increasing concentrations of imidazole (up to 250 mm) in 20 mm Tris/HCl, 500 mm NaCl and 0.2% Chaps (pH 8.0) for elution. Finally, decorin was separated via anion exchange chromatography on Q-Sepharose.
Following the endpoints of the specified treatments, MDA-MB-231 cells were briefly washed in ice-cold NaCl/Pi and lysed in RIPA buffer (1% Triton X-100, 20 mm Tris/HCl, pH 8.0, 137 mm NaCl, 10% glycerol, 2 mm EDTA, 1 mm Na3VO4, 10 μg·mL−1 aprotinin, 10 μg·mL−1 leupeptin) for 20 min on ice. Samples were resolved by SDS/PAGE on 8%, 10% or 12% gels before transfer to nitrocellulose for immunoblotting against the desired protein targets. Equivalent loading and normalization of the samples were confirmed following Coomassie Blue (CB) staining carried out on a nontransferred portion of the SDS/PAGE gel.
Slot blot assays for detection of secreted TSP-1
Slot blot analysis of secreted TSP-1 in MDA-MB-231 cells entailed treatment with 200 nm decorin protein core at the indicated time points, as required by the experimental condition, following 2 h of serum starvation. MDA-MD-231 cells were seeded at a density of ~ 3 × 105 cells in six-well dishes, and allowed to grow to ~ 85% confluency (~ 1 × 106 cells). Then, TCM was collected, centrifuged at 1000 g for 5 min, and passed through a disposable 0.22-μm syringe-driven filter unit (Millipore). Serial dilutions (maintaining a total volume of 400 μL) of the conditioned medium were applied to the sample acceptor of the slot blot apparatus with suction (60 mBar) for a minimum of 30 min, to ensure sample adsorption to the nitrocellulose membrane. The resulting membrane was blocked overnight in 1% BSA, and incubated with a 1 : 500 dilution of the primary antibody (mouse mAb against TSP-1), and then with a 1 : 6000 dilution of the secondary antibody [HRP-conjugated donkey anti-(mouse IgG)], with visualization via enhanced chemiluminescence on an ImageQuant LAS-4000 (GE Healthcare, USA). We quantified, via densitometry with the imagej program (NIH, Bethesda, MD, USA), the top band (as reported above), which represents a 1 : 2 dilution (200 μL of TCM), and the bottom band, which represents a 1 : 4 dilution (100 μL of TCM). These dilutions were done in a constant volume of 400 μL (DMEM was used as diluent) that was loaded into each slot and reacted with the antibody against TSP-1. Quantification of secreted TSP-1 bands was normalized to total cell number over time. The findings for the 1 : 2 and 1 : 4 dilutions were averaged and reported. This method was applied for all subsequent slot blot analyses.
Confocal microscopy, immunofluorescence imaging, and quantification
Confocal microscopy and immunofluorescence studies were performed as described previously [52, 92, 93]. Briefly, approximately 5 × 104 MDA-MB-231 cells were seeded on four-well glass chamber slides (BD Biosciences, San Jose, CA, USA), which had been previously coated with 0.2% gelatin. MDA-MB-231 cells were serum-starved (2 h) prior to incubation with decorin (200 nm). Cells were then washed with NaCl/Pi, fixed with 4% paraformaldehyde on ice for 20 min, blocked in NaCl/Pi and 1% BSA overnight at 4 °C, incubated with the appropriate primary antibodies (1 h) at room temperature, washed in NaCl/Pi, and then incubated in secondary antibody [Alexa-Fluor 488-conjugated goat anti-(rabbit IgG); Invitrogen] . The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA). Images were obtained with a × 63 1.3 oil-immersion objective on a Zeiss LSM-780 confocal laser scanning microscope (Zeiss, Oberkochen, Germany). The images were acquired with zen 2010 software with the filters set at 488 nm for imaging, and further analyzed with imagej and adobe photoshop CS5.1 (Adobe Systems, San Jose, CA, USA). For the immunofluorescence imaging studies, the slides were visualized with a Leica DM5500B microscope equipped with a Leica D-LUX3 camera in conjunction with advanced fluorescence 1.8 software (Leica Microsystems, New York City, NY, USA). Quantification of the relative fluorescence intensity of the TSP-1 signal was performed with imagej . Representative images were converted to black and white images with the split channel option. The background was then subtracted and the threshold adjusted so that only fluorescent particles remained. The total amount of fluorescent particles was then quantified with the analyze particle option in imagej.
siRNA-mediated silencing of ROCK1
Transient transfection with siRNA has been described elsewhere . Briefly, ROCK1 was silenced by use of of a cocktail composed of three validated siRNAs specific for ROCK1 mRNA (ROCK1 siRNA sc-29473; Santa Cruz Biotechnology). Six-well plates of MDA-MB-231 cells (containing ~ 2 × 105 cells) were transfected with either the siScramble control (sc-37007; Santa Cruz Biotechnology) at 20 pm or siROCK1 at 60 pm, with diluted Lipofectamine 2000 (Invitrogen) in transfection medium (1% BCS-DMEM) at ~ 70% confluency. The transfection was carried out for a total of 48 h at 37 °C, and was followed by treatment with decorin (200 nm) at the indicated time point. ROCK1 depletion was verified by immunoblotting with specific primary antibodies against ROCK1 prior to slot blot analysis of the TCM for TSP-1.
Exosomal isolation and purification
Exosome isolation and purification were carried as described elsewhere . Briefly, conditioned meda from four 10-cm dishes with ~ 4 × 107 MDA-MB-231 cells were collected. Cells were treated for 6 h with 200 nm decorin in 1% BCS-DMEM, and media were then pooled and centrifuged, initially at 2000 g for 15 min at 4 °C, and then at 4000 g for 15 min at 4 °C, in order to remove debris. Finally, exosomes were collected from the supernatant by ultracentrifugation at 105 000 g for 90 min at 4 °C. The resulting pellet was resuspended in NaCl/Pi, and subjected to immunoblotting analysis.
Each experiment was repeated three or more times, with similar patterns of response. All data were expressed as means ± standard errors of the mean (SEMs). The results were statistically analyzed with Student's t-test or a paired t-test, with sigma-stat 11.0 (SPSS). P < 0.05 was considered to be statistically significant.
We thank U. Rodeck (Kimmel Cancer Center, Thomas Jefferson University) for the generous gift of the monoclonal EGFR-blocking antibody, mAb425. This work was supported in part by National Institutes of Health grants RO1 CA39481, RO1 CA47282, and RO1 CA120975 (to R. V. Iozzo). T. Neill was supported by NIH training grant T32 AA07463, and this work is a part of T. Neill's doctoral thesis in Cell and Developmental Biology, Thomas Jefferson University.