TGFB1 is secreted through an unconventional pathway dependent on the autophagic machinery and cytoskeletal regulators

TGFB1 (transforming growth factor beta 1) is a potent cytokine playing a driving role in development, fibrosis and cancer. It is synthesized as prodomain-growth factor complex that requires tethering to LTBP (latent transforming growth factor beta binding protein) for efficient secretion into the extracellular space. Upon release, this large latent complex is sequestered by anchorage to extracellular matrix (ECM) networks, from which the mature growth factor needs to be activated in order to reach its receptors and initiate signaling. Here, we uncovered a novel intracellular secretion pathway by which the latent TGFB1 complex reaches the plasma membrane and is released from fibroblasts, the key effector cells during tissue repair, fibrosis and in the tumor stroma. We show that secretion of latent TGFB1, but not of other selected cytokines or of bulk cargo, is regulated by fibroblast-ECM communication through ILK (integrin linked kinase) that restricts RHOA activity by interacting with ARHGAP26/GRAF1. Latent TGFB1 interacts with GORASP2/GRASP55 and is detected inside MAP1LC3-positive autophagosomal intermediates that are secreted by a RAB8A-dependent pathway. Interestingly, TGFB1 secretion is fully abrogated in human and murine fibroblasts and macrophages that lack key components of the autophagic machinery. Our data demonstrate an unconventional secretion mode of TGFB1 adding another level of control of its bioavailability and activity in order to effectively orchestrate cellular programs prone to dysregulation as seen in fibrosis and cancer.     

Signaling through ShcA is required for transforming growth factor beta- and Neu/ErbB-2-induced breast cancer cell motility and invasion

Cooperation between the Neu/ErbB-2 and transforming growth factor beta (TGF-beta) signaling pathways enhances the invasive and metastatic capabilities of breast cancer cells; however, the underlying mechanisms mediating this synergy have yet to be fully explained. We demonstrate that TGF-beta induces the migration and invasion of mammary tumor explants expressing an activated Neu/ErbB-2 receptor, which requires signaling from autophosphorylation sites located in the C terminus. A systematic analysis of mammary tumor explants expressing Neu/ErbB-2 add-back receptors that couple to distinct signaling molecules has mapped the synergistic effect of TGF-beta-induced motility and invasion to signals emanating from tyrosine residues 1226/1227 and 1253 of Neu/ErbB-2. Given that the ShcA adaptor protein is known to interact with Neu/ErbB-2 through these residues, we investigated the importance of this signaling molecule in TGF-beta-induced cell motility and invasion. The reduction of ShcA expression rendered cells expressing activated Neu/ErbB-2, or add-back receptors signaling specifically through tyrosines 1226/1227 or 1253, unresponsive to TGF-beta-induced motility and invasion. In addition, a dominant-negative form of ShcA, lacking its three known tyrosine phosphorylation sites, completely abrogates the TGF-beta-induced migration and invasion of breast cancer cells expressing activated Neu/ErbB-2. Our results implicate signaling through the ShcA adaptor as a key component in the synergistic interaction between these pathways.     

Integrated genomics of chemotherapy resistant ovarian cancer: A role for extracellular matrix,TGFbeta and regulating microRNAs

Epithelial ovarian cancer is the sixth most common cancer in women worldwide and the most important cause of death from gynaecological cancers in the Western world.Our explorative pathway analysis on seven published gene-sets associated with platinum resistance in ovarian cancer reveals TP53 and transforming growth factor beta as key genes. Furthermore, the extracellular matrix was associated with chemotherapy resistance in ovarian cancer as well as endocrine resistance in breast cancer. Pathway analysis again revealed transforming growth factor beta as a key gene regulating extracellular matrix gene expression. A model is presented based on literature linking transforming growth factor beta, extracellular matrix, integrin signalling, epithelial to mesenchymal transition and regulating microRNAs with a (bivalent) role in chemotherapy response.     

TGF-β1 increases invasiveness of SW1990 cells through Rac1/ROS/NF-κB/IL-6/MMP-2

Human pancreatic cancer invasion and metastasis have been found to correlate with increased levels of active matrix metalloproteinase 2 (MMP-2). The multifunctional cytokine transforming growth factor beta 1 (TGF-β1) has been shown to increase both secretion of MMP-2 and invasion by several pancreatic cancer cell types. In the present study, we investigated the signaling pathway involved in TGF-β1-promoted MMP-2 secretion and invasion by human pancreatic cancer cells SW1990. Using specific inhibitors, we found that stimulation of these tumor cells with TGF-β1 induced secretion and activation of the collagenase MMP-2, which was required for TGF-β1-stimulated invasion. Our results also indicate that signaling events involved in TGF-β1-enhanced SW1990 invasiveness comprehend activation of Rac1 followed by generation of reactive oxygen species through nicotinamide adenine dinucleotide phosphate-oxidase, activation of nuclear factor-kappa beta, release of interleukin-6, and secretion and activation of MMP-2.     

ARF1 regulates the Rho/MLC pathway to control EGF-dependent breast cancer cell invasion

Invasion of tumor cells is a key step in metastasis that depends largely on the ability of these cells to degrade the extracellular matrix. Although we have showed that the GTPase ADP-ribosylation factor 1 (ARF1) is overexpressed in highly invasive breast cancer cell lines and that epidermal growth factor stimulation can activate this ARF isoform to regulate migration as well as proliferation, the role of this small GTP-binding protein has not been addressed in the context of invasiveness. Here we report that modulation of ARF1 expression and activity markedly impaired the ability of M.D. Anderson-metastatic breast-231 cells, a prototypical highly invasive breast cancer cell line, to degrade the extracellular matrix by controlling metalloproteinase-9 activity. In addition, we demonstrate that this occurs through inhibition of invadopodia maturation and shedding of membrane-derived microvesicles, the two key structures involved in invasion. To further define the molecular mechanisms by which ARF1 controls invasiveness, we show that ARF1 acts to modulate RhoA and RhoC activity, which in turn affects myosin light-chain (MLC) phosphorylation. Together our findings underscore for the first time a key role for ARF1 in invasion of breast cancer cells and suggest that targeting the ARF/Rho/MLC signaling axis might be a promising strategy to inhibit invasiveness and metastasis.     

Surface proteome analysis identifies platelet derived growth factor receptor-alpha as a critical mediator of transforming growth factor-beta-induced collagen secretion

Fibroblasts are extracellular matrix-producing cells in the lung. Fibroblast activation by transforming growth factor-beta leads to myofibroblast-differentiation and increased extracellular matrix deposition, a hallmark of pulmonary fibrosis. While fibroblast function with respect to migration, invasion, and extracellular matrix deposition has been well-explored, little is known about the surface proteome of lung fibroblasts in general and its specific response to fibrogenic growth factors, in particular transforming growth factor-beta.We thus performed a cell-surface proteome analysis of primary human lung fibroblasts in presence/absence of transforming growth factor-beta, followed by characterization of our findings using FACS analysis, Western blot, and siRNA-mediated knockdown experiments.We identified 213 surface proteins significantly regulated by transforming growth factor-beta, platelet derived growth factor receptor-alpha being one of the top down-regulated proteins. Transforming growth factor beta-induced downregulation of platelet derived growth factor receptor-alpha induced upregulation of platelet derived growth factor receptor-beta expression and phosphorylation of Akt, a downstream target of platelet derived growth factor signaling. Importantly, collagen type V expression and secretion was strongly increased after forced knockdown of platelet derived growth factor receptor-alpha, an effect that was potentiated by transforming growth factor-beta. We therefore show previously underappreciated cross-talk of transforming growth factor-beta and platelet derived growth factor signaling in human lung fibroblasts, resulting in increased extracellular matrix deposition in a platelet derived growth factor receptor-alpha dependent manner. These findings are of particular importance for the treatment of lung fibrosis patients with high pulmonary transforming growth factor-beta activity.     

Distinct mechanisms mediate the initial and sustained phases of cell migration in epidermal growth factor receptor-overexpressing cells

Elevated levels of epidermal growth factor receptor (EGFR) are predictive of increased invasion and metastasis in many human cancers. In the present study, we have shown that two distinct pathways regulate cell migration in EGFR-overexpressing invasive cells such as MDA 468 breast cancer cells: mitogen-activated protein kinase (MAPK or ERK 1 and 2) pathways play a major role in early stages to cell migration; and protein kinase C delta isoforms (PKC-delta) play a significant role in later stages of sustained cell migration. Inhibition of MAPK activity with MAP kinase kinase (MEK) inhibitor PD98059 blocks early stages of cell migration (up to 4 h); however, cells revert back to enhanced cell migration after 4 h. While inhibition of PKC-delta activity with rottlerin or dominant-negative PKC-delta expression blocks sustained cell migration after 4 h and up to 12 h, the combination of MAPK and PKC inhibitors completely blocked transforming growth factor alpha (TGF-alpha)-induced cell migration in EGFR-overexpressing breast cancer cells. However, inhibition of MAPK activity completely blocked cell migration in low EGFR-expressing non-invasive breast cancer cells such as MCF-7 cells. Forced overexpression of EGFR in MCF-7 cells (EGFR/MCF-7 cells) resulted in cell migration patterns seen in MDA 468 cells, that is, MAPK pathways play a major role in early stages to cell migration, and PKC-delta plays a major role in later stages of sustained cell migration. The above data demonstrate that EGFR-overexpressing invasive cells have the ability to compensate the loss of MAPK-mediated signaling through activation of PKC-delta signaling for cell migration, which plays a major role in invasion and metastasis. In addition, data suggest that inhibition of MAPK and PKC-delta signaling pathways should abrogate cell migration and invasion in EGFR-overexpressing human breast cancer cells.     

Epidermal growth factor-induced tumor cell invasion and metastasis initiated by dephosphorylation and downregulation of focal adhesion kinase

Upregulated epidermal growth factor (EGF) receptor (EGFR) expression and EGFR-induced signaling have been correlated with progression to invasion and metastasis in a wide variety of carcinomas, but the mechanism behind this is not well understood. We show here that, in various human carcinoma cells that overexpress EGFR, EGF treatment induced rapid tyrosine dephosphorylation of focal adhesion kinase (FAK) associated with downregulation of its kinase activity. The downregulation of FAK activity was both required and sufficient for EGF-induced refractile morphological changes, detachment of cells from the extracellular matrix, and increased tumor cell motility, invasion, and metastasis. Tumor cells with downregulated FAK activity became less adherent to the extracellular matrix. However, once cells started reattaching, FAK activity was restored by activated integrin signaling. Moreover, this process of readhesion and spreading could not be abrogated by further EGF stimulation. Interruption of transforming growth factor alpha-EGFR autocrine regulation with an EGFR tyrosine kinase inhibitor led to a substantial increase in FAK tyrosine phosphorylation and inhibition of tumor cell invasion in vitro. Consistent with this, FAK tyrosine phosphorylation was reduced in cells from tumors growing in transplanted, athymic, nude mice, which have an intact autocrine regulation of the EGFR. We suggest that the dynamic regulation of FAK activity, initiated by EGF-induced downregulation of FAK leading to cell detachment and increased motility and invasion, followed by integrin-dependent reactivation during readhesion, plays a role in EGF-associated tumor invasion and metastasis.     

Formation of atypical podosomes in extravillous trophoblasts regulates extracellular matrix degradation

Throughout pregnancy the cytotrophoblast, the stem cell of the placenta, gives rise to the differentiated forms of trophoblasts. The two main cell lineages are the syncytiotrophoblast and the invading extravillous trophoblast. A successful pregnancy requires extravillous trophoblasts to migrate and invade through the decidua and then remodel the maternal spiral arteries. Many invasive cells use specialised cellular structures called invadopodia or podosomes in order to degrade extracellular matrix. Despite being highly invasive cells, the presence of invadapodia or podosomes has not previously been investigated in trophoblasts. In this study these structures have been identified and characterised in extravillous trophoblasts. The role of specialised invasive structures in trophoblasts in the degradation of the extracellular matrix was compared with well characterised podosomes and invadopodia in other invasive cells and the trophoblast specific structures were characterised by using a sensitive matrix degradation assay which enabled visualisation of the structures and their dynamics. We show trophoblasts form actin rich protrusive structures which have the ability to degrade the extracellular matrix during invasion. The degradation ability and dynamics of the structures closely resemble podosomes, but have unique characteristics that have not previously been described in other cell types. The composition of these structures does not conform to the classic podosome structure, with no distinct ring of plaque proteins such as paxillin or vinculin. In addition, trophoblast podosomes protrude more deeply into the extracellular matrix than established podosomes, resembling invadopodia in this regard. We also show several significant pathways such as Src kinase, MAPK kinase and PKC along with MMP-2 and 9 as key regulators of extracellular matrix degradation activity in trophoblasts, while podosome activity was regulated by the rigidity of the extracellular matrix.     

Actopaxin (α-Parvin) Phosphorylation Is Required for Matrix Degradation and Cancer Cell Invasion

Dysregulation of cell adhesion and motility is known to be an important factor in the development of tumor malignancy. Actopaxin (α-parvin) is a paxillin, integrin-linked kinase, and F-actin binding focal adhesion protein with several serine phosphorylation sites in the amino terminus that contribute to the regulation of cell spreading and migration. Here, phosphorylation of actopaxin is shown to contribute to the regulation of matrix degradation and cell invasion. Osteosarcoma cells stably expressing wild type (WT), nonphosphorylatable (Quint), and phosphomimetic (S4D/S8D) actopaxin demonstrate that actopaxin phosphorylation is necessary for efficient Src and matrix metalloproteinase-driven degradation of extracellular matrix. Rac1 was found to be required for actopaxin-induced matrix degradation whereas inhibition of myosin contractility promoted degradation in the phosphomutant-expressing Quint cells, indicating that a balance of Rho GTPase signaling and regulation of cellular tension are important for the process. Furthermore, actopaxin forms a complex with the Rac1/Cdc42 GEF β-PIX and Rac1/Cdc42 effector PAK1, to regulate actopaxin-dependent matrix degradation. Actopaxin phosphorylation is elevated in the invasive breast cancer cell line MDA-MB-231 compared with normal breast epithelial MCF10A cells. Expression of the nonphosphorylatable Quint actopaxin in MDA-MB-231 cells inhibits cell invasion whereas overexpression of WT actopaxin promotes invasion in MCF10A cells. Taken together, this study demonstrates a new role for actopaxin phosphorylation in matrix degradation and cell invasion via regulation of Rho GTPase signaling.     

Preclinical Efficacy of Cystatin C to Target the Oncogenic Activity of Transforming Growth Factor β in Breast Cancer

We previously identified cystatin C (CystC) as a novel antagonist of transforming growth factor β (TGF-β) signaling in normal and malignant cells. However, whether the anti-TGF-β activities of CystC can be translated to preclinical animal models of breast cancer growth and metastasis remains unproven. Assessing the preclinical efficacy of CystC was accomplished using metastatic 4T1 breast cancer cells, whose oncogenic responses to TGF-β were inhibited both in vitro and in vivo. Indeed, we observed CystC to prevent TGF-β from stimulating the growth and pulmonary metastasis of 4T1 tumors in mice in part by reducing the extent of Smad2, p38 mitogen-activated protein kinase, and extracellular signal-regulated kinase 1/2 phosphorylation present in 4T1 tumors. We also found CystC to significantly antagonize angiogenesis in developing 4T1 tumors, suggesting a novel role for CystC in uncoupling TGF-β signaling in endothelial cells (ECs). Accordingly, CystC dramatically reduced murine and human EC responsiveness to TGF-β, including their ability to regulate the expression of 1) TGF-β signaling components, 2) inhibitor of differentiation (ID) family members, and 3) matrix metalloproteinases and their inhibitors (TIMPs) and to undergo cell invasion and angiogenic sprouting stimulated by TGF-β. Importantly, CystC prevented TGF-β from stimulating vessel development in Matrigel plugs implanted into genetically normal mice. Collectively, our findings provide the first preclinical evidence that CystC is efficacious in preventing breast cancer progression and angiogenesis stimulated by the oncogenic TGF-β signaling system and suggest that CystC-based chemotherapeutics possesses translational efficacy to one day treat and improve the clinical course of late-stage breast cancers.     

ABL Tyrosine Kinase Inhibition Variable Effects on the Invasive Properties of Different Triple Negative Breast Cancer Cell Lines

The non-receptor tyrosine kinase ABL drives myeloid progenitor expansion in human chronic myeloid leukemia. ABL inhibition by the tyrosine kinase inhibitor nilotinib is a first-line treatment for this disease. Recently, ABL has also been implicated in the transforming properties of solid tumors, including triple negative (TN) breast cancer. TN breast cancers are highly metastatic and several cell lines derived from these tumors display high invasive activity in vitro. This feature is associated with the activation of actin-rich membrane structures called invadopodia that promote extracellular matrix degradation. Here, we investigated nilotinib effect on the invasive and migratory properties of different TN breast cancer cell lines. Nilotinib decreased both matrix degradation and invasion in the TN breast cancer cell lines MDA-MB 231 and MDA-MB 468. However, and unexpectedly, nilotinib increased by two-fold the invasive properties of the TN breast cancer cell line BT-549 and of Src-transformed fibroblasts. Both display much higher levels of ABL kinase activity compared to MDA-MB 231. Similar effects were obtained by siRNA-mediated down-regulation of ABL expression, confirming ABL central role in this process. ABL anti-tumor effect in BT-549 cells and Src-transformed fibroblasts was not dependent on EGF secretion, as recently reported in neck and squamous carcinoma cells. Rather, we identified the TRIO-RAC1 axis as an important downstream element of ABL activity in these cancer cells. In conclusion, the observation that TN breast cancer cell lines respond differently to ABL inhibitors could have implications for future therapies.     

Multi-step pericellular proteolysis controls the transition from individual to collective cancer cell invasion

Invasive cell migration through tissue barriers requires pericellular remodelling of extracellular matrix (ECM) executed by cell-surface proteases, particularly membrane-type-1 matrix metalloproteinase (MT1-MMP/MMP-14). Using time-resolved multimodal microscopy, we show how invasive HT-1080 fibrosarcoma and MDA-MB-231 breast cancer cells coordinate mechanotransduction and fibrillar collagen remodelling by segregating the anterior force-generating leading edge containing beta1 integrin, MT1-MMP and F-actin from a posterior proteolytic zone executing fibre breakdown. During forward movement, sterically impeding fibres are selectively realigned into microtracks of single-cell calibre. Microtracks become expanded by multiple following cells by means of the large-scale degradation of lateral ECM interfaces, ultimately prompting transition towards collective invasion similar to that in vivo. Both ECM track widening and transition to multicellular invasion are dependent on MT1-MMP-mediated collagenolysis, shown by broad-spectrum protease inhibition and RNA interference. Thus, invasive migration and proteolytic ECM remodelling are interdependent processes that control tissue micropatterning and macropatterning and, consequently, individual and collective cell migration.     

Dual role of SnoN in mammalian tumorigenesis

SnoN is an important negative regulator of transforming growth factor beta signaling through its ability to interact with and repress the activity of Smad proteins. It was originally identified as an oncoprotein based on its ability to induce anchorage-independent growth in chicken embryo fibroblasts. However, the roles of SnoN in mammalian epithelial carcinogenesis have not been well defined. Here we show for the first time that SnoN plays an important but complex role in human cancer. SnoN expression is highly elevated in many human cancer cell lines, and this high level of SnoN promotes mitogenic transformation of breast and lung cancer cell lines in vitro and tumor growth in vivo, consistent with its proposed pro-oncogenic role. However, this high level of SnoN expression also inhibits epithelial-to-mesenchymal transdifferentiation. Breast and lung cancer cells expressing the shRNA for SnoN exhibited an increase in cell motility, actin stress fiber formation, metalloprotease activity, and extracellular matrix production as well as a reduction in adherens junction proteins. Supporting this observation, in an in vivo breast cancer metastasis model, reducing SnoN expression was found to moderately enhance metastasis of human breast cancer cells to bone and lung. Thus, SnoN plays both pro-tumorigenic and antitumorigenic roles at different stages of mammalian malignant progression. The growth-promoting activity of SnoN appears to require its ability to bind to and repress the Smad proteins, while the antitumorigenic activity can be mediated by both Smad-dependent and Smad-independent pathways and requires the activity of small GTPase RhoA. Our study has established the importance of SnoN in mammalian epithelial carcinogenesis and revealed a novel aspect of SnoN function in malignant progression.     

MicroRNA-7 Inhibits Epithelial-to-Mesenchymal Transition and Metastasis of Breast Cancer Cells via Targeting FAK Expression

Focal adhesion kinase (FAK) is an important mediator of extracellular matrix integrin signaling, cell motility, cell proliferation and cell survival. Increased FAK expression is observed in a variety of solid human tumors and increased FAK expression and activity frequently correlate with metastatic disease and poor prognosis. Herein we identify miR-7 as a direct regulator of FAK expression. miR-7 expression is decreased in malignant versus normal breast tissue and its expression correlates inversely with metastasis in human breast cancer patients. Forced expression of miR-7 produced increased E-CADHERIN and decreased FIBRONECTIN and VIMENTIN expression in breast cancer cells. The levels of miR-7 expression was positively correlated with E-CADHERIN mRNA and negatively correlated with VIMENTIN mRNA levels in breast cancer samples. Forced expression of miR-7 in aggressive breast cancer cell lines suppressed tumor cell monolayer proliferation, anchorage independent growth, three-dimensional growth in Matrigel, migration and invasion. Conversely, inhibition of miR-7 in the HBL-100 mammary epithelial cell line promoted cell proliferation and anchorage independent growth. Rescue of FAK expression reversed miR-7 suppression of migration and invasion. miR-7 also inhibited primary breast tumor development, local invasion and metastatic colonization of breast cancer xenografts. Thus, miR-7 expression is decreased in metastatic breast cancer, correlates with the level of epithelial differentiation of the tumor and inhibits metastatic progression.     

Adipose tissue-derived stem cells differentiate into carcinoma-associated fibroblast-like cells under the influence of tumor-derived factors

Carcinoma-associated fibroblasts (CAF) are considered to contribute to tumor growth, invasion and metastasis. However, the cell type of origin remains unknown. Since human adipose tissue-derived stem cells (hASCs) are locally adjacent to breast cancer cells and might directly interact with tumor cells, we investigated whether CAFs may originate from hASCs. We demonstrated that a significant percentage of hASCs differentiated into a CAF-like myofibroblastic phenotype (e.g., expression of alpha smooth muscle actin and tenascin-C) when exposed to conditioned medium from the human breast cancer lines MDAMB231 and MCF7. The conditioned medium from MDAMB231 and MCF7 contains significant amounts of transforming growth factor-beta 1 (TGFβ1) and the differentiation of hASCs towards CAFs is dependent on TGFβ1 signaling via Smad3 in hASCs. The induction of CAFs can be abolished using a neutralizing antibody to TGFβ1 as well as by pretreatment of the hASCs with SB431542, a TGFβ1 receptor kinase inhibitor. Additionally, we found that these hASC-derived CAF-like cells exhibit functional properties of CAFs, including the ability to promote tumor cell invasion in an in vitro invasion assay, as well as increased expression of stromal-cell-derived factor 1 (SDF-1) and CCL5. Taken together, these data suggest that hASCs are a source of CAFs which play an important role in the tumor invasion.