Breast cancer is second only to lung cancer in cancer-related deaths in women, and the majority of these deaths are caused by metastases. Obtaining a better understanding of migration and invasion, ...two early steps in metastasis, is critical for the development of treatments that inhibit breast cancer metastasis. In a functional proteomic screen for proteins required for invasion, extracellular heat shock protein 90 alpha (Hsp90α) was identified and shown to activate matrix metalloproteinase 2 (MMP-2). The mechanism of MMP-2 activation by Hsp90α is unknown. Intracellular Hsp90α commonly functions with a complex of co-chaperones, leading to our hypothesis that Hsp90α functions similarly outside of the cell. In this study, we show that a complex of co-chaperones outside of breast cancer cells assists Hsp90α mediated activation of MMP-2. We demonstrate that the co-chaperones Hsp70, Hop, Hsp40, and p23 are present outside of breast cancer cells and co-immunoprecipitate with Hsp90α in vitro and in breast cancer conditioned media. These co-chaperones also increase the association of Hsp90α and MMP-2 in vitro. This co-chaperone complex enhances Hsp90α-mediated activation of MMP-2 in vitro, while inhibition of Hsp70 in conditioned media reduces this activation and decreases cancer cell migration and invasion. Together, these findings support a model in which MMP-2 activation by an extracellular co-chaperone complex mediated by Hsp90α increases breast cancer cell migration and invasion. Our studies provide insight into a novel pathway for MMP-2 activation and suggest Hsp70 as an additional extracellular target for anti-metastatic drug development.
Abstract
Background
Metastasis is a multi-step process that is responsible for the majority of deaths in cancer patients. Current treatments are not effective in targeting metastasis. The molecular ...chaperone hsp90α is secreted from invasive cancer cells and activates MMP-2 to enhance invasiveness, required for the first step in metastasis.
Methods
We analyzed the morphology and motility of invasive cancer cells that were treated with exogenous exosomes in the presence or absence of hsp90α. We performed mass spectrometry and immunoprecipitation to identify plasminogen as a potential client protein of extracellular hsp90α. Plasmin activation assays and migration assays were performed to test if plasminogen is activated by extracellular hsp90α and has a role in migration.
Results
We found that hsp90α is secreted in exosomes in invasive cancer cells and it contributes to their invasive nature. We identified a novel interaction between hsp90α and tissue plasminogen activator that together with annexin II, also found in exosomes, activates plasmin. Extracellular hsp90α promotes plasmin activation as well as increases plasmin dependent cell motility.
Conclusions
Our data indicate that hsp90α is released by invasive cancer cells via exosomes and implicates hsp90α in activating plasmin, a second protease that acts in cancer cell invasion.
Obesity is one of the most important preventable causes of cancer and the most significant risk factor for breast cancer in postmenopausal women. Compared with lean women, obese women are more likely ...to be diagnosed with a larger, higher grade tumor, an increased incidence of lymph node metastases, and elevated risk of distant recurrence. However, the mechanisms connecting obesity to the pathogenesis of breast cancer are poorly defined. Here, we show that during obesity, adipocytes within human and mouse breast tissues recruit and activate macrophages through a previously uncharacterized CCL2/IL-1β/CXCL12 signaling pathway. Activated macrophages in turn promote stromal vascularization and angiogenesis even before the formation of cancer. Recapitulating these changes using a novel humanized breast cancer model was sufficient to promote angiogenesis and prime the microenvironment prior to neoplastic transformation for accelerated breast oncogenesis. These findings provide a mechanistic role for adipocytes and macrophages before carcinogenesis that may be critical for prevention and treatment of obesity-related cancer.
Pregnancy associated breast cancers (PABCs) exhibit increased aggressiveness and overall poorer survival. During lactation, changes take place in the breast tissue microenvironment that lead to ...increased macrophage recruitment and alterations in adipose stromal cells (ASC-Ls). The interaction of these cells in PABCs could play a role in the increased aggressiveness of these cancers. We utilized an in vitro co-culture model to recreate the interactions of ASC-Ls and macrophages in vivo. We performed qRT-PCR to observe changes in gene expression and cytokine arrays to identify transcriptional changes that result in an altered microenvironment. Additionally, functional assays were performed to further elicit how these changes affect tumorigenesis. The co-culture of ASC-Ls and macrophages altered both mRNA expression and cytokine secretion in a tumor promoting manner. Tumorigenic cytokines, such as IL-6, CXCL1, CXCL5, and MMP-9 secretion levels, were enhanced in the co-culture. Additionally, conditioned media from the co-culture elevated the tumor cell proliferation and angiogenic potential of endothelial cells. These finds indicate that the changes seen in the microenvironment of PABC, specifically the secretion of cytokines, play a role in the increased tumorigenesis of PABCs by altering the microenvironment to become more favorable to tumor progression.
Obesity is correlated with worsened prognosis and treatment resistance in breast cancer. Macrophage-targeted therapies are currently in clinical trials, however, little is known about how obesity may ...impact treatment efficacy. Within breast adipose tissue, obesity leads to chronic, macrophage-driven inflammation, suggesting that obese breast cancer patients may benefit from these therapies. Using a high fat diet model of obesity, we orthotopically transplanted cancer cell lines into the mammary glands of obese and lean mice. We quantified changes in tumor invasiveness, angiogenesis and metastasis, and examined the efficacy of macrophage depletion to diminish tumor progression in obese and lean mice. Mammary tumors from obese mice grew significantly faster, were enriched for cancer stem-like cells (CSCs) and were more locally invasive and metastatic. Tumor cells isolated from obese mice demonstrated enhanced expression of stem cell-related pathways including
and
. Despite more rapid growth, mammary tumors from obese mice had reduced necrosis, higher blood vessel density, and greater macrophage recruitment. Depletion of macrophages in obese tumor-bearing mice resulted in increased tumor necrosis, reduced endothelial cells, and enhanced recruitment of CD8
T cells compared to IgG-treated controls. Macrophages may be an important clinical target to improve treatment options for obese breast cancer patients.
Estrogen has a central role in the genesis and progression of breast cancers whether they are positive or negative for the estrogen receptor (ER). While therapies that disrupt estrogen biosynthesis ...or ER activity can treat these diseases in postmenopausal women, in younger women where ovarian function remains intact, these anti-estrogen therapies are not as effective. Moreover, emerging clinical evidence suggests that estrogen may promote other cancers. Thus, circulating estrogens may participate in cancer pathogenesis in ways that are not yet understood. In this study, we show that estrogen can promote the outgrowth of murine xenograft tumors established from patient-derived ER-negative breast cancer cells by influencing the mobilization and recruitment of a proangiogenic population of bone marrow-derived myeloid cells. ERα expression was necessary and sufficient in the bone marrow-derived cells themselves to promote tumor formation in response to estrogen. Our findings reveal a novel way in which estrogen promotes tumor formation, with implications for the development and application of anti-estrogen therapies to treat cancer in premenopausal women.
Extracellular Hsp90 (eHsp90) activates a number of client proteins outside of cancer cells required for migration and invasion. Therefore, eHsp90 may serve as a novel target for anti-metastatic drugs ...as its inhibition using impermeant Hsp90 inhibitors would not affect the numerous vital intracellular Hsp90 functions in normal cells. While some eHsp90 clients are known, it is important to establish other proteins that act outside the cell to validate eHsp90 as a drug target to limit cancer spread. Using mass spectrometry we identified two precursor proteins Galectin 3 binding protein (G3BP) and Lysyl oxidase 2-like protein (LOXL2) that associate with eHsp90 in MDA-MB231 breast cancer cell conditioned media and confirmed that LOXL2 binds to eHsp90 in immunoprecipitates. We introduce a novel impermeant Hsp90 inhibitor STA-12-7191 derived from ganetespib and show that it is markedly less toxic to cells and can inhibit cancer cell migration in a dose dependent manner. We used STA-12-7191 to test if LOXL2 and G3BP are potential eHsp90 clients. We showed that while LOXL2 can increase wound healing and compensate for STA-12-7191-mediated inhibition of wound closure, addition of G3BP had no affect on this assay. These findings support of role for LOXL2 in eHsp90 stimulated cancer cell migration and provide preliminary evidence for the use of STA-12-7191 to inhibit eHsp90 to limit cancer invasion.
Oesophageal adenocarcinoma (OAC) is an exemplar model of obesity-associated cancer. Previous work in our group has demonstrated that overweight/obese OAC patients have better responses to neoadjuvant ...therapy, but the underlying mechanisms are unknown. Unravelling the immune–metabolic signatures of adipose tissue may provide insight for this observation. We hypothesised that different metabolic pathways predominate in visceral (VAT) and subcutaneous adipose tissue (SAT) and inflammatory secretions will differ between the fat depots. Real-time ex vivo metabolic profiles of VAT and SAT from 12 OAC patients were analysed. These samples were screened for the secretion of 54 inflammatory mediators, and data were correlated with patient body composition. Oxidative phosphorylation (OXPHOS) was significantly higher in VAT when compared to SAT. OXPHOS was significantly higher in the SAT of patients receiving neoadjuvant treatment. VEGF-A, VEGF-C, P1GF, Flt-1, bFGF, IL-15, IL-16, IL-17A, CRP, SAA, ICAM-1, VCAM-1, IL-2, IL-13, IFN-γ, and MIP-1β secretions were significantly higher from VAT than SAT. Higher levels of bFGF, Eotaxin-3, and TNF-α were secreted from the VAT of obese patients, while higher levels of IL-23 and TARC were secreted from the SAT of obese patients. The angiogenic factors, bFGF and VEGF-C, correlated with visceral fat area. Levels of OXPHOS are higher in VAT than SAT. Angiogenic, vascular injury and inflammatory cytokines are elevated in VAT versus SAT, indicating that VAT may promote inflammation, linked to regulating treatment response.
Abstract
Obesity is a growing health concern worldwide and increases the incidence of multiple types of cancer, including breast cancer. Obese breast cancer patients often develop more aggressive ...tumors than lean patients and have increased risk for metastasis, tumor recurrence and mortality. Here, we sought to address how obesity alters the biology of breast cancer to promote aggressive tumors. To induce obesity, we fed mice either a control diet (CD) or high fat diet (HFD) for 16 weeks, then transplanted Met-1 tumor cells into mammary fat pads and monitored tumor growth. At end stage, tumors from HFD-fed mice were significantly larger than tumors from CD-fed mice, suggesting obesity promotes tumor growth. To investigate how obesity promotes tumor aggression, we dissociated the tumors from CD- and HFD-fed mice and plated isolated tumor cells in tumorsphere and invasion assays to test for cells with cancer stem-like cell (CSC) properties. Tumor cells from HFD-fed mice demonstrated increased tumorsphere formation and increased capacity for invasion compared to tumor cells from CD-fed mice, suggesting that obesity selects for tumor cells with CSC properties. Next, to address how obesity impacts the tumor microenvironment, we evaluated tumor necrosis and blood vessel formation through CD31 staining. Tumors from HFD-fed mice had significantly less necrosis and greater CD31 staining than those from CD-fed mice, suggesting that obesity promotes tumor angiogenesis. Since obesity promotes chronic, macrophage-driven inflammation within adipose tissue of the mammary gland, we stained tumors for the macrophage marker, F4/80. As with obese mammary glands, tumors from HFD-fed mice had significantly greater macrophage recruitment than tumors from CD-fed mice, together suggesting that obesity alters the tumor microenvironment. To determine how obesity stimulates tumor angiogenesis, we performed an in vitro assay by culturing dissociated tumor cells from HFD or CD-fed mice alone or with macrophages. Conditioned media (CM) isolated from tumor cells from HFD-fed mice cultured with macrophages enhanced the ability of endothelial cells to form networks in vitro. In contrast, CM from HFD tumor cells alone, macrophages alone, or those from CD-fed mice did not promote network formation. Together, these results suggest that cooperation between macrophages and tumor cells from HFD-fed mice promotes angiogenesis. Next, to investigate how macrophages and tumor cells interacting in obesity, we depleted macrophages using anti-F4/80 antibodies in CD-fed and HFD-fed tumor-bearing mice. In HFD-fed mice, macrophage depletion significantly reduced tumor volume and CD31 staining while increasing tumor necrosis compared to controls. Obesity promotes interactions between tumor cells and macrophages to enhance tumor angiogenesis and progression.
Breast cancer is second only to lung cancer in cancer-related deaths in women, and the majority of these deaths are caused by metastases. Obtaining a better understanding of migration and invasion, ...two early steps in metastasis, is critical for the development of treatments that inhibit breast cancer metastasis. In a functional proteomic screen for proteins required for invasion, extracellular heat shock protein 90 alpha (Hsp90alpha) was identified and shown to activate matrix metalloproteinase 2 (MMP-2). The mechanism of MMP-2 activation by Hsp90alpha is unknown. Intracellular Hsp90alpha commonly functions with a complex of co-chaperones, leading to our hypothesis that Hsp90alpha functions similarly outside of the cell. In this study, we show that a complex of co-chaperones outside of breast cancer cells assists Hsp90alpha mediated activation of MMP-2. We demonstrate that the co-chaperones Hsp70, Hop, Hsp40, and p23 are present outside of breast cancer cells and co-immunoprecipitate with Hsp90alpha in vitro and in breast cancer conditioned media. These co-chaperones also increase the association of Hsp90alpha and MMP-2 in vitro. This co-chaperone complex enhances Hsp90alpha-mediated activation of MMP-2 in vitro, while inhibition of Hsp70 in conditioned media reduces this activation and decreases cancer cell migration and invasion. Together, these findings support a model in which MMP-2 activation by an extracellular co-chaperone complex mediated by Hsp90alpha increases breast cancer cell migration and invasion. Our studies provide insight into a novel pathway for MMP-2 activation and suggest Hsp70 as an additional extracellular target for anti-metastatic drug development.
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